Small organic molecule regulators of cell proliferation

ABSTRACT

A compound represented in general formula (X) and (XI):

RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.09/724,492, filed Nov. 28, 2000, which is based on U.S. ProvisionalApplication No. 60/193,279, filed Mar. 30, 2000, the specifications ofwhich is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Pattern formation is the activity by which embryonic cells form orderedspatial arrangements of differentiated tissues. The physical complexityof higher organisms arises during embryogenesis through the interplay ofcell-intrinsic lineage and cell-extrinsic signaling. Inductiveinteractions are essential to embryonic patterning in vertebratedevelopment from the earliest establishment of the body plan, to thepatterning of the organ systems, to the generation of diverse cell typesduring tissue differentiation (Davidson, E., (1990) Development 108:365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al.,(1992) Cell 68: 257-270). The effects of developmental cell interactionsare varied. Typically, responding cells are diverted from one route ofcell differentiation to another by inducing cells that differ from boththe uninduced and induced states of the responding cells (inductions).Sometimes cells induce their neighbors to differentiate like themselves(homeogenetic induction); in other cases a cell inhibits its neighborsfrom differentiating like itself. Cell interactions in early developmentmay be sequential, such that an initial induction between two cell typesleads to a progressive amplification of diversity. Moreover, inductiveinteractions occur not only in embryos, but in adult cells as well, andcan act to establish and maintain morphogenetic patterns as well asinduce differentiation (J. B. Gurdon (1992) Cell 68:185-199).

Members of the Hedgehog family of signaling molecules mediate manyimportant short- and long-range patterning processes during invertebrateand vertebrate development. In the fly, a single hedgehog gene regulatessegmental and imaginal disc patterning. In contrast, in vertebrates, ahedgehog gene family is involved in the control of left-right asymmetry,polarity in the CNS, somites and limb, organogenesis, chondrogenesis andspermatogenesis.

The first hedgehog gene was identified by a genetic screen in the fruitfly Drosophila melanogaster (Nüsslein-Volhard, C. and Wieschaus, E.(1980) Nature 287, 795-801). This screen identified a number ofmutations affecting embryonic and larval development. In 1992 and 1993,the molecular nature of the Drosophila hedgehog (hh) gene was reported(C. F., Lee et al. (1992) Cell 71, 33-50), and since then, severalhedgehog homologues have been isolated from various vertebrate species.While only one hedgehog gene has been found in Drosophila and otherinvertebrates, multiple Hedgehog genes are present in vertebrates.

The vertebrate family of hedgehog genes includes at least four members,e.g., paralogs of the single drosophila hedgehog gene. Exemplaryhedgehog genes and proteins are described in PCT publications WO95/18856 and WO 96/17924. Three of these members, herein referred to asDesert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh),apparently exist in all vertebrates, including fish, birds, and mammals.A fourth member, herein referred to as tiggie-winkle hedgehog (Thh),appears specific to fish. Desert hedgehog (Dhh) is expressed principallyin the testes, both in mouse embryonic development and in the adultrodent and human; Indian hedgehog (Ihh) is involved in bone developmentduring embryogenesis and in bone formation in the adult; and, Shh, whichas described above, is primarily involved in morphogenic andneuroinductive activities. Given the critical inductive roles ofhedgehog polypeptides in the development and maintenance of vertebrateorgans, the identification of hedgehog interacting proteins is ofparamount significance in both clinical and research contexts.

The various Hedgehog proteins consist of a signal peptide, a highlyconserved N-terminal region, and a more divergent C-terminal domain. Inaddition to signal sequence cleavage in the secretory pathway (Lee, J.J. et al. (1992) Cell 71:33-50; Tabata, T. et al. (1992) Genes Dev.2635-2645; Chang, D. E. et al. (1994) Development 120:3339-3353),Hedgehog precursor proteins undergo an internal autoproteolytic cleavagewhich depends on conserved sequences in the C-terminal portion (Lee etal. (1994) Science 266:1528-1537; Porter et al. (1995) Nature374:363-366). This autocleavage leads to a 19 kD N-terminal peptide anda C-terminal peptide of 26-28 kD (Lee et al. (1992) supra; Tabata et al.(1992) supra; Chang et al. (1994) supra; Lee et al. (1994) supra;Bumcrot, D. A., et al. (1995) Mol. Cell. Biol. 15:2294-2303; Porter etal. (1995) supra; Ekker, S.C. et al. (1995) Curr. Biol. 5:944-955; Lai,C. J. et al. (1995) Development 121:2349-2360). The N-terminal peptidestays tightly associated with the surface of cells in which it wassynthesized, while the C-terminal peptide is freely diffusible both invitro and in vivo (Porter et al. (1995) Nature 374:363; Lee et al.(1994) supra; Bumcrot et al. (1995) supra; Mart', E. et al. (1995)Development 121:2537-2547; Roelink, H. et al. (1995) Cell 81:445-455).Interestingly, cell surface retention of the N-terminal peptide isdependent on autocleavage, as a truncated form of HH encoded by an RNAwhich terminates precisely at the normal position of internal cleavageis diffusible in vitro (Porter et al. (1995) supra) and in vivo (Porter,J. A. et al. (1996) Cell 86, 21-34). Biochemical studies have shown thatthe autoproteolytic cleavage of the HH precursor protein proceedsthrough an internal thioester intermediate which subsequently is cleavedin a nucleophilic substitution. It is likely that the nucleophile is asmall lipophilic molecule which becomes covalently bound to theC-terminal end of the N-peptide (Porter et al. (1996) supra), tetheringit to the cell surface. The biological implications are profound. As aresult of the tethering, a high local concentration of N-terminalHedgehog peptide is generated on the surface of the Hedgehog producingcells. It is this N-terminal peptide which is both necessary andsufficient for short- and long-range Hedgehog signaling activities inDrosophila and vertebrates (Porter et al. (1995) supra; Ekker et al.(1995) supra; Lai et al. (1995) supra; Roelink, H. et al. (1995) Cell81:445-455; Porter et al. (1996) supra; Fietz, M. J. et al. (1995) Curr.Biol. 5:643-651; Fan, C.-M. et al. (1995) Cell 81:457-465; Mart', E., etal. (1995) Nature 375:322-325; Lopez-Martinez et al. (1995) Curr. Biol5:791-795; Ekker, S. C. et al. (1995) Development 121:2337-2347; Forbes,A. J. et al. (1996) Development 122:1125-1135).

HH has been implicated in short- and long-range patterning processes atvarious sites during Drosophila development. In the establishment ofsegment polarity in early embryos, it has short-range effects whichappear to be directly mediated, while in the patterning of the imaginaldiscs, it induces long-range effects via the induction of secondarysignals.

In vertebrates, several hedgehog genes have been cloned in the past fewyears. Of these genes, Shh has received most of the experimentalattention, as it is expressed in different organizing centers which arethe sources of signals that pattern neighboring tissues. Recent evidenceindicates that Shh is involved in these interactions.

The expression of Shh starts shortly after the onset of gastrulation inthe presumptive midline mesoderm, the node in the mouse (Chang et al.(1994) supra; Echelard, Y. et al. (1993) Cell 75:1417-1430), the rat(Roelink, H. et al. (1994) Cell 76:761-775) and the chick (Riddle, R. D.et al. (1993) Cell 75:1401-1416), and the shield in the zebrafish (Ekkeret al. (1995) supra; Krauss, S. et al.(1993) Cell 75:1431-1444). Inchick embryos, the Shh expression pattern in the node develops aleft-right asymmetry, which appears to be responsible for the left-rightsitus of the heart (Levin, M. et al. (1995) Cell 82:803-814).

In the CNS, Shh from the notochord and the floorplate appears to induceventral cell fates. When ectopically expressed, Shh leads to aventralization of large regions of the mid- and hindbrain in mouse(Echelard et al. (1993) supra; Goodrich, L. V. et al. (1996) Genes Dev.10:301-312), Xenopus (Roelink, H. et al. (1994) supra; Ruiz i Altaba, A.et al. (1995) Mol. Cell. Neurosci. 6:106-121), and zebrafish (Ekker etal. (1995) supra; Krauss et al. (1993) supra; Hammerschmidt, M., et al.(1996) Genes Dev. 10:647-658). In explants of intermediate neuroectodermat spinal cord levels, Shh protein induces floorplate and motor neurondevelopment with distinct concentration thresholds, floor plate at highand motor neurons at lower concentrations (Roelink et al. (1995) supra;Mart' et al. (1995) supra; Tanabe, Y. et al. (1995) Curr. Biol.5:651-658). Moreover, antibody blocking suggests that Shh produced bythe notochord is required for notochord-mediated induction of motorneuron fates (Mart' et al. (1995) supra). Thus, high concentration ofShh on the surface of Shh-producing midline cells appears to account forthe contact-mediated induction of floorplate observed in vitro (Placzek,M. et al. (1993) Development 117:205-218), and the midline positioningof the floorplate immediately above the notochord in vivo. Lowerconcentrations of Shh released from the notochord and the floorplatepresumably induce motor neurons at more distant ventrolateral regions ina process that has been shown to be contact-independent in vitro(Yamada, T. et al. (1993) Cell 73:673-686). In explants taken atmidbrain and forebrain levels, Shh also induces the appropriateventrolateral neuronal cell types, dopaminergic (Heynes, M. et al.(1995) Neuron 15:35-44; Wang, M. Z. et al. (1995) Nature Med.1:1184-1188) and cholinergic (Ericson, J. et al. (1995) Cell 81:747-756)precursors, respectively, indicating that Shh is a common inducer ofventral specification over the entire length of the CNS. Theseobservations raise a question as to how the differential response to Shhis regulated at particular anteroposterior positions.

Shh from the midline also patterns the paraxial regions of thevertebrate embryo, the somites in the trunk (Fan et al. (1995) supra)and the head mesenchyme rostral of the somites (Hammerschmidt et al.(1996) supra). In chick and mouse paraxial mesoderm explants, Shhpromotes the expression of sclerotome-specific markers like Pax1 andTwist, at the expense of the dermamyotomal marker Pax3. Moreover, filterbarrier experiments suggest that Shh mediates the induction of thesclerotome directly rather than by activation of a secondary signalingmechanism (Fan, C.-M. and Tessier-Lavigne, M. (1994) Cell 79,1175-1186).

Shh also induces myotomal gene expression (Hammerschmidt et al. (1996)supra; Johnson, R. L. et al. (1994) Cell 79:1165-1173; Münsterberg, A.E. et al. (1995) Genes Dev. 9:2911-2922; Weinberg, E. S. et al. (1996)Development 122:271-280), although recent experiments indicate thatmembers of the WNT family, vertebrate homologues of Drosophila wingless,are required in concert (Münsterberg et al. (1995) supra). Puzzlingly,myotomal induction in chicks requires higher Shh concentrations than theinduction of sclerotomal markers (Münsterberg et al. (1995) supra),although the sclerotome originates from somitic cells positioned muchcloser to the notochord. Similar results were obtained in the zebrafish,where high concentrations of Hedgehog induce myotomal and represssclerotomal marker gene expression (Hammerschmidt et al. (1996) supra).In contrast to amniotes, however, these observations are consistent withthe architecture of the fish embryo, as here, the myotome is thepredominant and more axial component of the somites. Thus, modulation ofShh signaling and the acquisition of new signaling factors may havemodified the somite structure during vertebrate evolution.

In the vertebrate limb buds, a subset of posterior mesenchymal cells,the “Zone of polarizing activity” (ZPA), regulates anteroposterior digitidentity (reviewed in Honig, L. S. (1981) Nature 291:72-73). Ectopicexpression of Shh or application of beads soaked in Shh peptide mimicsthe effect of anterior ZPA grafts, generating a mirror image duplicationof digits (Chang et al. (1994) supra; Lopez-Martinez et al. (1995)supra; Riddle et al. (1993) supra) (FIG. 2g). Thus, digit identityappears to depend primarily on Shh concentration, although it ispossible that other signals may relay this information over thesubstantial distances that appear to be required for AP patterning(100-150 μm). Similar to the interaction of HH and DPP in the Drosophilaimaginal discs, Shh in the vertebrate limb bud activates the expressionof Bmp2 (Francis, P. H. et al. (1994) Development 120:209-218), a dpphomologue. However, unlike DPP in Drosophila, Bmp2 fails to mimic thepolarizing effect of Shh upon ectopic application in the chick limb bud(Francis et al. (1994) supra). In addition to anteroposteriorpatterning, Shh also appears to be involved in the regulation of theproximodistal outgrowth of the limbs by inducing the synthesis of thefibroblast growth factor FGF4 in the posterior apical ectodermal ridge(Laufer, E. et al. (1994) Cell 79:993-1003; Niswander, L. et al.(1994)Nature 371:609-612).

The close relationship between Hedgehog proteins and BMPs is likely tohave been conserved at many, but probably not all sites of vertebrateHedgehog expression. For example, in the chick hindgut, Shh has beenshown to induce the expression of Bmp4, another vertebrate dpp homologue(Roberts, D. J. et al. (1995) Development 121:3163-3174). Furthermore,Shh and Bmp2, 4, or 6 show a striking correlation in their expression inepithelial and mesenchymal cells of the stomach, the urogenital system,the lung, the tooth buds and the hair follicles (Bitgood, M. J. andMcMahon, A. P. (1995) Dev. Biol. 172:126-138). Further, Ihh, one of thetwo other mouse Hedgehog genes, is expressed adjacent to Bmp expressingcells in the gut and developing cartilage (Bitgood and McMahon (1995)supra).

Recent evidence suggests a model in which Ihh plays a crucial role inthe regulation of chondrogenic development (Roberts et al. (1995)supra). During cartilage formation, chondrocytes proceed from aproliferating state via an intermediate, prehypertrophic state todifferentiated hypertrophic chondrocytes. Ihh is expressed in theprehypertrophic chondrocytes and initiates a signaling cascade thatleads to the blockage of chondrocyte differentiation. Its direct targetis the perichondrium around the Ihh expression domain, which responds bythe expression of Gli and Patched (Ptc), conserved transcriptionaltargets of Hedgehog signals (see below). Most likely, this leads tosecondary signaling resulting in the synthesis of parathyroidhormone-related protein (PTHrP) in the periarticular perichondrium.PTHrP itself signals back to the prehypertrophic chondrocytes, blockingtheir further differentiation. At the same time, PTHrP repressesexpression of Ihh, thereby forming a negative feedback loop thatmodulates the rate of chondrocyte differentiation.

Patched was originally identified in Drosophila as a segment polaritygene, one of a group of developmental genes that affect celldifferentiation within the individual segments that occur in ahomologous series along the anterior-posterior axis of the embryo. SeeHooper, J. E. et al. (1989) Cell 59:751; and Nakano, Y. et al. (1989)Nature 341:508. Patterns of expression of the vertebrate homologue ofpatched suggest its involvement in the development of neural tube,skeleton, limbs, craniofacial structure, and skin.

Genetic and functional studies demonstrate that patched is part of thehedgehog signaling cascade, an evolutionarily conserved pathway thatregulates expression of a number of downstream genes. See Perrimon, N.(1995) Cell 80:517; and Perrimon, N. (1996) Cell 86:513. Patchedparticipates in the constitutive transcriptional repression of thetarget genes; its effect is opposed by a secreted glycoprotein, encodedby hedgehog, or a vertebrate homologue, which induces transcriptionalactivation. Genes under control of this pathway include members of theWnt and TGF-beta families.

Patched proteins possess two large extracellular domains, twelvetransmembrane segments, and several cytoplasmic segments. See Hooper,supra; Nakano, supra; Johnson, R. L. et al. (1996) Science 272:1668; andHahn, H. et al. (1996) Cell 85:841. The biochemical role of patched inthe hedgehog signalling pathway is unclear. Direct interaction with thehedgehog protein has, however, been reported (Chen, Y. et al. (1996)Cell 87:553), and patched may participate in a hedgehog receptor complexalong with another transmembrane protein encoded by the smoothened gene.See Perrimon, supra; and Chen, supra.

The human homologue of patched was recently cloned and mapped tochromosome 9q22.3. See Johnson, supra; and Hahn, supra. This region hasbeen implicated in basal cell nevus syndrome (BCNS), which ischaracterized by developmental abnormalities including rib andcraniofacial alterations, abnormalities of the hands and feet, and spinabifida.

BCNS also predisposes to multiple tumor types, the most frequent beingbasal cell carcinomas (BCC) that occur in many locations on the body andappear within the first two decades of life. Most cases of BCC, however,are unrelated to the syndrome and arise sporadically in small numbers onsun-exposed sites of middle-aged or older people of northern Europeanancestry.

Recent studies in BCNS-related and sporadic BCC suggest that afunctional loss of both alleles of patched leads to development of BCC.See Johnson, supra; Hahn, supra; and Gailani, M. R. et al. (1996) NatureGenetics 14:78. Single allele deletions of chromosome 9q22.3 occurfrequently in both sporadic and hereditary BCC. Linkage analysisrevealed that the defective inherited allele was retained and the normalallele was lost in tumors from BCNS patients.

Sporadic tumors also demonstrated a loss of both functional alleles ofpatched. Of twelve tumors in which patched mutations were identifiedwith a single strand conformational polymorphism screening assay, ninehad chromosomal deletion of the second allele and the other three hadinactivating mutations in both alleles (Gailani, supra). The alterationsdid not occur in the corresponding germline DNA.

Most of the identified mutations resulted in premature stop codons orframe shifts. Lench, N. J., et al., Hum. Genet. October 1997; 100(5-6):497-502. Several, however, were point mutations leading to amino acidsubstitutions in either extracellular or cytoplasmic domains. Thesesites of mutation may indicate functional importance for interactionwith extracellular proteins or with cytoplasmic members of thedownstream signaling pathway.

The involvement of patched in the inhibition of gene expression and theoccurrence of frequent allelic deletions of patched in BCC support atumor suppressor function for this gene. Its role in the regulation ofgene families known to be involved in cell signaling and intercellularcommunication provides a possible mechanism of tumor suppression.

SUMMARY OF THE INVENTION

The present invention makes available methods and compositions formodulating differentiation or proliferation of a cell. Compounds usefulin such methods and compositions include those represented by generalformula (I):

wherein, as valence and stability permit,

Ar and Ar′ independently represent substituted or unsubstituted aryl orheteroaryl rings;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

Cy and Cy′ independenly represent substituted or unsubstituted aryl,heterocyclyl, heteroaryl, or cycloalkyl, including polycyclic groups;and

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Ar and Ar′ represent phenyl rings, e.g.,unsubstituted or substituted with one or more groups includingheteroatoms such as O, N, and S. In certain embodiments, at least one ofAr and Ar′ represents a phenyl ring. In certain embodiments, at leastone of Ar and Ar′ represents a heteroaryl ring, e.g., a pyridyl,thiazolyl, thienyl, pyrimidyl, etc. In certain embodiments, Y and Ar′are attached to Ar in a meta and/or 1,3-relationship.

In certain embodiments, Y is absent from all positions. In embodimentswherein Y is present in a position, i preferably represents an integerfrom 1-2 in an adjacent M_(i) if i=0 would result in two occurrences ofY being directly attached, or an occurrence of Y being directly attachedto N.

In certain embodiments, Cy′ is a substituted or unsubstituted aryl orheteroaryl. In certain embodiments, Cy′ is directly attached to X. Incertain embodiments, Cy′ is a substituted or unsubstituted bicyclic orheteroaryl ring, preferably both bicyclic and heteroaryl, such asbenzothiophene, benzofuran, benzopyrrole, benzopyridine, etc. In certainembodiments, Cy′ is a monocyclic aryl or heteroaryl ring substituted atleast with a substituted or unsubstituted aryl or heteroaryl ring, i.e.,forming a biaryl system. In certain embodiments, Cy′ includes twosubstituted or unsubstituted aryl or heteroaryl rings, e.g., the same ordifferent, directly connected by one or more bonds, e.g., to form abiaryl or bicyclic ring system.

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, R represents H or lower alkyl, e.g., H or Me.

In certain embodiments, Cy represents a substituted or unsubstitutednon-aromatic carbocyclic or heterocyclic ring, i.e., including at leastone sp³ hybridized atom, and preferably a plurality of sp³ hybridizedatoms. In certain embodiments, Cy includes an amine within the atoms ofthe ring or on a substituent of the ring, e.g., Cy is pyridyl,imidazolyl, pyrrolyl, piperidyl, pyrrolidyl, piperazyl, etc., and/orbears an amino substituent. In certain embodiments, Cy is a 5- to7-membered ring. In certain embodiments, Cy is directly attached to N.In embodiments wherein Cy is a six-membered ring directly attached to Nand bears an amino substituent at the 4 position of the ring relative toN, the N and amine substituents may be disposed trans on the ring.

In certain embodiments, substituents on Ar or Ar′ are selected fromhalogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove, wherein p and n, individually for each occurrence, representintegers from 0 to 10, preferably from 0 to 5.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (II):

wherein, as valence and stability permit,

Ar and Ar′ independently represent substituted or unsubstituted aryl orheteroaryl rings;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—;

X is selected from —C(═O)—, —C(═S)—, —S(O2)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH2—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne, wherein some or all occurrences of M inM_(j) form all or part of a cyclic structure;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

Cy′ represents a substituted or unsubstituted aryl, heterocyclyl,heteroaryl, or cycloalkyl, including polycyclic groups;

j represents, independently for each occurrence, an integer from 0 to10, preferably from 2 to 7; and

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Ar and Ar′ represent phenyl rings, e.g.,unsubstituted or substituted with one or more groups includingheteroatoms such as O, N, and S. In certain embodiments, at least one ofAr and Ar′ represents a phenyl ring. In certain embodiments, at leastone of Ar and Ar′ represents a heteroaryl ring, e.g., a pyridyl,thiazolyl, thienyl, pyrimidyl, etc. In certain embodiments, Y and Ar′are attached to Ar in a meta and/or 1,3-relationship.

In certain embodiments, Y is absent from all positions. In embodimentswherein Y is present in a position, i preferably represents an integerfrom 1-2 in an adjacent M_(i) if i=0 would result in two occurrences ofY being directly attached, or an occurrence of Y being directly attachedto N or NR₂.

In certain embodiments, Cy′ is a substituted or unsubstituted aryl orheteroaryl. In certain embodiments, Cy′ is directly attached to X. Incertain embodiments, Cy′ is a substituted or unsubstituted bicyclic orheteroaryl ring, preferably both bicyclic and heteroaryl, such asbenzothiophene, benzofuran, benzopyrrole, benzopyridine, etc. In certainembodiments, Cy′ is a monocyclic aryl or heteroaryl ring substituted atleast with a substituted or unsubstituted aryl or heteroaryl ring, i.e.,forming a biaryl system. In certain embodiments, Cy′ includes twosubstituted or unsubstituted aryl or heteroaryl rings, e.g., the same ordifferent, directly connected by one or more bonds, e.g., to form abiaryl or bicyclic ring system.

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, R represents H or lower alkyl, e.g., H or Me.

In certain embodiments, NR₂ represents a primary amine or a secondary ortertiary amine substituted with one or two lower alkyl groups, arylgroups, or aralkyl groups, respectively, preferably a primary amine orsecondary amine.

In certain embodiments, substituents on Ar or Ar′ are selected fromhalogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove, wherein p and n, individually for each occurrence, representintegers from 0 to 10, preferably from 0 to 5.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (III):

wherein, as valence and stability permit,

Ar and Ar′ independently represent substituted or unsubstituted aryl orheteroaryl rings;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

Cy and Cy′ independenly represent substituted or unsubstituted aryl,heterocyclyl, heteroaryl, or cycloalkyl, including polycyclic groups;and

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Ar and Ar′ represent phenyl rings, e.g.,unsubstituted or substituted with one or more groups includingheteroatoms such as O, N, and S. In certain embodiments, at least one ofAr and Ar′ represents a phenyl ring. In certain embodiments, at leastone of Ar and Ar′ represents a heteroaryl ring, e.g., a pyridyl,thiazolyl, thienyl, pyrimidyl, etc. In certain embodiments, Y and Ar′are attached to Ar in a meta and/or 1,3-relationship.

In certain embodiments, Y is absent from all positions. In embodimentswherein Y is present in a position, i preferably represents an integerfrom 1-2 in an adjacent M_(i) if i=0 would result in two occurrences ofY being directly attached, or an occurrence of Y being directly attachedto N or NR₂.

In certain embodiments, Cy′ is a substituted or unsubstituted aryl orheteroaryl. In certain embodiments, Cy′ is directly attached to X. Incertain embodiments, Cy′ is a substituted or unsubstituted bicyclic orheteroaryl ring, preferably both bicyclic and heteroaryl, such asbenzothiophene, benzofuran, benzopyrrole, benzopyridine, etc. In certainembodiments, Cy′ is a monocyclic aryl or heteroaryl ring substituted atleast with a substituted or unsubstituted aryl or heteroaryl ring, i.e.,forming a biaryl system. In certain embodiments, Cy′ includes twosubstituted or unsubstituted aryl or heteroaryl rings, e.g., the same ordifferent, directly connected by one or more bonds, e.g., to form abiaryl or bicyclic ring system.

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, R represents H or lower alkyl, e.g., H or Me.

In certain embodiments, NR₂ represents a primary amine or a secondary ortertiary amine substituted with one or two lower alkyl groups, arylgroups, or aralkyl groups, respectively, preferably a primary amine or asecondary amine.

In certain embodiments, Cy represents a substituted or unsubstitutednon-aromatic carbocyclic or heterocyclic ring, i.e., including at leastone sp³ hybridized atom, and preferably a plurality of sp³ hybridizedatoms. In certain embodiments, Cy is directly attached to N and/or toNR₂. In certain embodiments, Cy is a 5- to 7-membered ring. Inembodiments wherein Cy is a six-membered ring directly attached to N andbears an amino substituent at the 4 position of the ring relative to N,the N and amine substituents may be disposed trans on the ring.

In certain embodiments, substituents on Ar or Ar′ are selected fromhalogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove, wherein n and p, individually for each occurrence, representintegers from 0 to 10, preferably from 0 to 5.

In certain embodiments, compounds useful in the subject methods includecompounds represented by general formula (IV):

wherein, as valence and stability permit,

Cy′ represents a substituted or unsubstituted aryl or heteroaryl ring,including polycyclics;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

R₁ and R₂ represent, independently and as valency permits, from 0-5substituents on the ring to which it is attached, selected from halogen,lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl,ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove;

Cy represents substituted or unsubstituted aryl, heterocyclyl,heteroaryl, or cycloalkyl, including polycyclic groups;

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2; and

p and n, individually for each occurrence, represent integers from 0 to10, preferably from 0 to 5.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Cy′ represents a substituted or unsubstitutedbicyclic or heterocyclic ring system, preferably both bicyclic andheteroaryl, such as benzothiophene, benzofuran, benzopyrrole,benzopyridine, etc. In certain embodiments, Cy′ is directly attached toX. In certain embodiments, Cy′ is a monocyclic aryl or heteroaryl ringsubstituted at least with a substituted or unsubstituted aryl orheteroaryl ring, i.e., forming a biaryl system. In certain embodiments,Cy′ includes two substituted or unsubstituted aryl or heteroaryl rings,e.g., the same or different, directly connected by one or more bonds,e.g., to form a biaryl or bicyclic ring system.

In certain embodiments, Y is absent from all positions. In embodimentswherein Y is present in a position, i preferably represents an integerfrom 1-2 in an adjacent M_(i) if i=0 would result in two occurrences ofY being directly attached, or an occurrence of Y being directly attachedto N.

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, R represents H or lower alkyl, e.g., H or Me.

In certain embodiments, Cy represents a substituted or unsubstitutednon-aromatic carbocyclic or heterocyclic ring, i.e., including at leastone sp³ hybridized atom, and preferably a plurality of sp³ hybridizedatoms. In certain embodiments, Cy includes an amine within the atoms ofthe ring or on a substituent of the ring, e.g., Cy is pyridyl,imidazolyl, pyrrolyl, piperidyl, pyrrolidyl, piperazyl, etc., and/orbears an amino substituent. In certain embodiments, Cy is directlyattached to N. In certain embodiments, Cy is a 5- to 7-membered ring. Inembodiments wherein Cy is a six-membered ring directly attached to N andbears an amino substituent at the 4 position of the ring relative to N,the N and amine substituents may be disposed trans on the ring.

In certain embodiments, R₁ and R₂ represent, independently and asvalency permits, from 0-5 substituents on the ring to which it isattached, selected from halogen, lower alkyl, lower alkenyl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, —(CH₂)_(p)alkyl, —(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (V):

wherein, as valence and stability permit,

Cy′ represents a substituted or unsubstituted aryl or heteroaryl ring,including polycyclics;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

R₁ and R₂ represent, independently and as valency permits, from 0-5substituents on the ring to which it is attached, selected from halogen,lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl,ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove;

Cy′ represents a substituted or unsubstituted aryl, heterocyclyl,heteroaryl, or cycloalkyl, including polycyclic groups;

j represents, independently for each occurrence, an integer from 0 to10, preferably from 2 to 7;

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2; and

p and n, individually for each occurrence, represent integers from 0 to10, preferably from 0 to 5.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Cy′ represents a substituted or unsubstitutedbicyclic or heterocyclic ring system, preferably both bicyclic andheteroaryl, such as benzothiophene, benzofuran, benzopyrrole,benzopyridine, etc. In certain embodiments, Cy′ is directly attached toX. In certain embodiments, Cy′ is a monocyclic aryl or heteroaryl ringsubstituted at least with a substituted or unsubstituted aryl orheteroaryl ring, i.e., forming a biaryl system. In certain embodiments,Cy′ includes two substituted or unsubstituted aryl or heteroaryl rings,e.g., the same or different, directly connected by one or more bonds,e.g., to form a biaryl or bicyclic ring system.

In certain embodiments, Y is absent from all positions. In embodimentswherein Y is present in a position, i preferably represents an integerfrom 1-2 in an adjacent M_(i) if i=0 would result in two occurrences ofY being directly attached, or an occurrence of Y being directly attachedto N or NR₂.

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, NR₂ represents a primary amine or a secondary ortertiary amine substituted with one or two lower alkyl groups, arylgroups, or aralkyl groups, respectively, preferably a primary orsecondary amine.

In certain embodiments, R represents H or lower alkyl, e.g., H or Me.

In certain embodiments, R₁ and R₂ represent, independently and asvalency permits, from 0-5 substituents on the ring to which it isattached, selected from halogen, lower alkyl, lower alkenyl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, —(CH₂)_(p)alkyl, —(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (VI):

wherein, as valence and stability permit,

Cy′ represents a substituted or unsubstituted aryl or heteroaryl ring,including polycyclics;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

Cy represents substituted or unsubstituted aryl, heterocyclyl,heteroaryl, or cycloalkyl, including polycyclic groups;

R₁ and R₂ represent, independently and as valency permits, from 0-5substituents on the ring to which it is attached, selected from halogen,lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl,ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove;

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2; and

n and p, individually for each occurrence, represent integers from 0 to10, preferably from 0 to 5.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Cy′ represents a substituted or unsubstitutedbicyclic or heteroaryl ring system, preferably both bicyclic andheteroaryl, e.g., benzothiophene, benzofuran, benzopyrrole,benzopyridyl, etc. In certain embodiments, Cy′ is directly attached toX. In certain embodiments, Cy′ is a monocyclic aryl or heteroaryl ringsubstituted at least with a substituted or unsubstituted aryl orheteroaryl ring, i.e., forming a biaryl system. In certain embodiments,Cy′ includes two substituted or unsubstituted aryl or heteroaryl rings,e.g., the same or different, directly connected by one or more bonds,e.g., to form a biaryl or bicyclic ring system.

In certain embodiments, Y is absent from all positions. In embodimentswherein Y is present in a position, i preferably represents an integerfrom 1-2 in an adjacent M_(i) if i=0 would result in two occurrences ofY being directly attached, or an occurrence of Y being directly attachedto N or NR₂.

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, NR₂ represents a primary amine or a secondary ortertiary amine substituted with one or two lower alkyl groups, arylgroups, or aralkyl groups, respectively, preferably a primary amine.

In certain embodiments, R represents H or lower alkyl, e.g., H or Me.

In certain embodiments, Cy represents a substituted or unsubstitutednon-aromatic carbocyclic or heterocyclic ring, i.e., including at leastone sp³ hybridized atom, and preferably a plurality of sp³ hybridizedatoms. In certain embodiments, Cy is directly attached to N and/or toNR₂. In certain embodiments, Cy is a 5- to 7-membered ring. Inembodiments wherein Cy is a six-membered ring directly attached to N andbears an amino substituent at the 4 position of the ring relative to N,the N and amine substituents may be disposed trans on the ring.

In certain embodiments, R₁ and R₂ represent, independently and asvalency permits, from 0-5 substituents on the ring to which it isattached, selected from halogen, lower alkyl, lower alkenyl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, —(CH₂)_(p)alkyl, —(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove.

In certain embodiments, a subject compound has the structure of FormulaVII:

wherein, as valence and stability permit,

Cy represents a substituted or unsubstituted heterocyclyl or cycloalkyl;

Cy′ is a substituted or unsubstituted aryl or heteroaryl ring, includingpolycyclics;

W is O or S;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

R₁ and R₂ represent, independently and as valency permits, from 0-5substituents on the ring to which it is attached, selected from halogen,lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl,ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove;

n and p, individually for each occurrence, represent integers from 0 to10, preferably from 0 to 5.

In certain embodiments, Cy′ represents a substituted or unsubstitutedbicyclic or heteroaryl ring system, preferably both bicyclic andheteroaryl, e.g., benzothiophene, benzofuran, benzopyrrole,benzopyridyl, etc. In certain other embodiments, Cy′ represents an arylor heteroaryl ring substituted at least with a substituted orunsubstituted aryl or heteroaryl ring, i.e., to form a biaryl ringsystem.

In certain embodiments, NR₂ represents a primary amine or a secondary ortertiary amine substituted with one or two lower alkyl groups, arylgroups, or aralkyl groups, respectively, preferably a primary orsecondary amine.

In certain embodiments, Cy represents a substituted or unsubstitutedsaturated carbocyclic or heterocyclic ring, i.e., composed of aplurality of sp³ hybridized atoms. In certain embodiments, Cy is a 5- to7-membered ring. In embodiments wherein Cy is a six-membered ringdirectly attached to N and bears an amino substituent at the 4 positionof the ring relative to N, the N and amine substituents may be disposedtrans on the ring.

In certain embodiments, R₁ and R₂ represent, independently and asvalency permits, from 0-5 substituents on the ring to which it isattached, selected from halogen, lower alkyl, lower alkenyl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, —(CH₂)_(p)alkyl, —(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove.

In certain embodiments, a subject compound has a structure of FormulaVIII:

wherein, as valence and stability permit,

U represents a substituted or unsubstituted aryl or heteroaryl ringfused to the nitrogen-containing ring;

V represents a lower alkylene group, such as methylene, 1,2-ethylene,1,1-ethylene, 1,1-propylene, 1,2-propylene, 1,3-propylene, etc.;

W represents S or O, preferably O;

X represents C═O, C═S, or SO₂;

R₃ represents substituted or unsubstituted aryl, heteroaryl, loweralkyl, lower alkenyl, lower alkynyl, carbocyclyl, carbocyclylalkyl,heterocyclyl, heterocyclylalkyl, aralkyl, or heteroaralkyl;

R₄ represents substituted or unsubstituted aralkyl or lower alkyl, suchas phenethyl, benzyl, or aminoalkyl, etc.;

R₅ represents substituted or unsubstituted aryl, heteroaryl, aralkyl, orheteroaralkyl, including polycyclic aromatic or heteroaromatic groups.

In certain embodiments, U represents a phenyl ring fused to thenitrogen-containing ring.

In certain embodiments, R₃ is selected from substituted or unsubstitutedaryl, heteroaryl, lower alkyl, lower alkenyl, aralkyl, andheteroaralkyl.

In certain embodiments, R₄ is an unsubstituted lower alkyl group, or isa lower alkyl group substituted with a secondary or tertiary amine.

In certain embodiments, R₅ is selected from substituted or unsubstitutedphenyl or naphthyl, or is a diarylalkyl group, such as2,2-diphenylethyl, diphenylmethyl, etc.

Additionally, compounds having structures of Formulae IX-XII may beuseful in the compositions and methods of the present invention.

In certain embodiments, a hedgehog-independent compound useful in thepresent invention, such as described above, may have an EC₅₀ forinducing or augmenting one or more hedgehog activities (such asupregulation of gli expression) of less than about 1000 nm, less thanabout 100 nm, less than about 10 nm, or even less than about 1 nm. Incertain embodiments, a hedgehog-dependent compound useful in the presentinvention, such as described above, augmenting one or more hedgehogactivities (such as upregulation of gli expression) by at least one,two, or even three orders of magnitude.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-31 depict reactions useful for synthesizing compounds accordingto the present invention.

FIGS. 32 and 33 illustrate representative compounds according to thepresent invention.

FIGS. 34a and 34 b depict results from hedgehog pathway reporter assaysusing compounds of the present invention.

FIG. 35 presents assay results demonstrating upregulation of Ptc and Gliby hedgehog agonists of the present invention.

FIGS. 36 and 37 portray the increased proliferation of cerebellar neuronprecursors in the presence of subject agonists.

FIGS. 38A and B show the effect of subject agonists on healing of acrushed sciatic nerve as measured in a grip assay.

FIGS. 39A-D demonstrate the effect of subject agonists on healing of acrushed sciatic nerve as measured in a toe spread assay.

FIG. 40 shows the effect of subject agonists in combination with a lowdose of hedgehog protein on lung, scapula, skin, and skill tissue ofdeveloping mice.

FIG. 41 depicts the effect of subject agonists in with and without addedhedgehog protein on pancreas, kidney, skin, and heart tissue ofdeveloping mice.

FIG. 42 exhibits the effect of a subject agonist on the forelimb of anewborn mouse.

FIG. 43 presents the effect of a subject agonist on forelimbs of anewborn mouse at different concentrations.

FIG. 44 portrays the effects of a subject agonist on developing lungtissue.

FIG. 45 shows the effects of a subject agonist on developing kidneytissue.

FIGS. 46A and B shows the effects of subject agonists on mouse skintissue in the presence and absence of hedgehog protein.

FIGS. 47A and B compare the activity of subject agonists in mouse andhuman reporter cells.

FIG. 48 depicts the upregulation of Gli in HEPM cells treated with amodified N-terminal fragment of Sonic hedgehog or with a subjectagonist.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview

The present invention relates to the discovery that signal transductionpathways regulated by hedgehog, patched (ptc), gli and/or smoothened canbe modulated, at least in part, by small molecules. While not wishing tobe bound by any particular theory, the activation of apatched-smoothened pathway through alteration of cell-surfaceassociations (such as complexes) may be the mechanism by which theseagents act. The hedgehog pathway is believed to be negatively regulatedby association of patched and smoothened, such as in the form of proteincomplexes, which association is disrupted by the binding of hedgehog topatched. Accordingly, the ability of these agents to activate thehedgehog pathway may be due to the ability of such molecules to interactwith or bind to patched or smoothened, to otherwise disrupt theassociation of smoothened with patched, or at least to promote theability of those proteins to activate a hedgehog, ptc, and/orsmoothened-mediated signal transduction pathway. This mode of action,e.g., modulation of a smoothened-dependent pathway, is to bedistinguished from compounds which modulate the hedgehog pathway bydirectly activating the cAMP pathway, e.g., by binding to or interactingwith PKA, adenylate cyclase, cAMP phosphodiesterase, etc.

Certain hedgehog agonists disclosed herein modulate hedgehog activity inthe absence of hedgehog protein itself, e.g., the compounds mimic theactivity of hedgehog, rather than merely supplement or increase theactivity of hedgehog protein, e.g., by promoting hedgehog binding topatched. These compounds are referred to herein as hedgehog-independentagonists and alone can mimic the phenotype or effect resulting fromhedgehog treatment. Certain other compounds of the present inventionenhance the activity of hedgehog protein, and require the presence oraddition of hedgehog protein to observe the phenotype or effectresulting from hedgehog induction. Such hedgehog-dependent agonists maybe used in therapeutic preparations or treatments which include hedgehogprotein, or may be used to increase the activity of hedgehog proteinnaturally produced by the cells or tissue to be treated with theagonist. The hedgehog agonists disclosed herein may induce dissociationof a patched-smoothened complex or disrupt interactions between patchedand smoothened, such as by binding to patched or to smoothened, therebyactivating the hedgehog pathway. In certain embodiments, thecompositions and methods of the present invention employ a compoundwhich acts on one or more components of the extracellular membrane of atarget cell.

In certain embodiments, hedgehog agonists useful in the present inducehedgehog-dependent transcriptional regulation, such as expression of thegli1 or ptc genes. Such agonists can thus induce or increase thehedgehog-dependent pathway activation resulting from, for example,increased levels of hedgehog protein.

It is, therefore, specifically contemplated that these small moleculeswhich modulate aspects of hedgehog, ptc, or smoothened signaltransduction activity will likewise be capable of promotingproliferation (or other biological consequences) in cells having afunctional ptc-smo pathway. In preferred embodiments, the subjectagonists are organic molecules having a molecular weight less than 2500amu, more preferably less than 1500 amu, and even more preferably lessthan 750 amu, and are capable of inducing or augmenting at least some ofthe biological activities of hedgehog proteins, preferably specificallyin target cells. Activation of the hedgehog pathway by a hedgehogagonist may be quantified, for example, by detecting the increase in ptcor gli-1 transcription in the presence of the agonist relative to acontrol in the absence of agonist. For example, an increase of at least5%, at least 10%, at least 20%, or even at least 50% may be indicativeof hedgehog pathway activation by a test compound. In certainembodiments, the agonist activity of the subject compounds is notinhibited by the hedgehog antibody 5E1, but is inhibited by jervine oran antagonist having the formula:

This quality can be quantified, for example, by determining whether theantibody or antagonist induces a decrease of more than 50%, more than20%, more than 10%, or even more than 5% of the ptc or gli-1upregulation induced by the agonist in the absence of the hedgehogantagonist, etc. In certain embodiments, a compound useful in thepresent invention, such as described above, may have an EC₅₀ forinducing or augmenting one or more hedgehog activities (such asupregulation of ptc or gli expression) of less than about 1000 nM, lessthan about 100 nM, less than about 10 nM, or even less than about 1 nM.The coding sequences for exemplary human Gli genes include, for example,the Gli-1 gene sequence of GenBank accession X07384 and the Gli-2 genesequence of GenBank accession AB007298. See also Kinzler et al. Nature1988, 332, 371. The level of gli or ptc expression can be determined,for example, by measuring the level of mRNA (transcription) or the levelof protein (translation).

Thus, the methods of the present invention include the use of smallmolecules which antagonize ptc inhibition of hedgehog signalling, suchas by activating smoothened or downstream components of the signalpathway, in the regulation of repair and/or functional performance of awide range of cells, tissues and organs. For instance, the subjectmethod has therapeutic and cosmetic applications ranging from regulationof neural tissues, bone and cartilage formation and repair, regulationof spermatogenesis, regulation of smooth muscle, regulation of lung,liver, urogenital organs (e.g., bladder), and other organs arising fromthe primitive gut, regulation of hematopoietic function, regulation ofskin and hair growth, etc. Moreover, the subject methods can beperformed on cells which are provided in culture (in vitro), or on cellsin a whole animal (in vivo). See, for example, PCT publications WO95/18856 and WO 96/17924 (the specifications of which are expresslyincorporated by reference herein).

In one embodiment, the subject method can be to treat epithelial cells.In general, an epithelial cell may be contacted with an amount of ahedgehog agonist to induce epithelial tissue growth and/or formation.The subject method can be carried out on epithelial cells which may beeither dispersed in culture or a part of an intact tissue or organ.Moreover, the method can be performed on cells which are provided inculture (in vitro), or on cells in a whole animal (in vivo).

The hedgehog agonists of the present invention may be used as part ofregimens in the treatment of disorders of, or surgical or cosmeticrepair of, such epithelial tissues as skin and skin organs; corneal,lens and other ocular tissue; mucosal membranes; and periodontalepithelium. The methods and compositions disclosed herein provide forthe treatment or prevention of a variety of damaged epithelial andmucosal tissues. For instance, the subject method can be used to controlwound healing processes, as for example may be desirable in connectionwith any surgery involving epithelial tissue, such as fromdermatological or periodontal surgeries. Exemplary surgical repair forwhich hedgehog agonists may be useful include severe burn and skinregeneration, skin grafts, pressure sores, dermal ulcers, fissures, postsurgery scar reduction, and ulcerative colitis.

In another aspect of the present invention, a hedgehog agonist can beused to effect the growth of hair, as for example in the treatment ofalopecia whereby hair growth is potentiated.

In another preferred embodiment, the subject method can be used as partof a treatment regimen for malignant medulloblastoma and other primaryCNS malignant neuroectodermal tumors.

In another aspect, the present invention provides pharmaceuticalpreparations comprising, as an active ingredient, a hedgehog agonist,ptc antagonist, or smoothened agonist such as described herein,formulated in an amount sufficient to promote, in vivo, proliferation orother biological consequences.

The subject treatments using hedgehog agonists, patched antagonists, orsmoothened agonists can be effective for both human and animal subjects.Animal subjects to which the invention is applicable extend to bothdomestic animals and livestock, raised either as pets or for commercialpurposes. Examples are dogs, cats, cattle, horses, sheep, hogs, andgoats.

II. Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The phrase “aberrant modification or mutation” of a gene refers to suchgenetic lesions as, for example, deletions, substitution or addition ofnucleotides to a gene, as well as gross chromosomal rearrangements ofthe gene and/or abnormal methylation of the gene. Likewise,mis-expression of a gene refers to aberrant levels of transcription ofthe gene relative to those levels in a normal cell under similarconditions, as well as non-wild-type splicing of mRNA transcribed fromthe gene.

“Burn wounds” refer to cases where large surface areas of skin have beenremoved or lost from an individual due to heat and/or chemical agents.

The “corium” or “dermis” refers to the layer of the skin deep to theepidermis, consisting of a dense bed of vascular connective tissue, andcontaining the nerves and terminal organs of sensation. The hair roots,and sebaceous and sweat glands are structures of the epidermis which aredeeply embedded in the dermis.

“Dental tissue” refers to tissue in the mouth which is similar toepithelial tissue, for example gum tissue. The method of the presentinvention is useful for treating periodontal disease.

“Dermal skin ulcers” refer to lesions on the skin caused by superficialloss of tissue, usually with inflammation. Dermal skin ulcers which canbe treated by the method of the present invention include decubitusulcers, diabetic ulcers, venous stasis ulcers and arterial ulcers.Decubitus wounds refer to chronic ulcers that result from pressureapplied to areas of the skin for extended periods of time. Wounds ofthis type are often called bedsores or pressure sores. Venous stasisulcers result from the stagnation of blood or other fluids fromdefective veins. Arterial ulcers refer to necrotic skin in the areaaround arteries having poor blood flow.

The term “ED₅₀” means the dose of a drug which produces 50% of itsmaximum response or effect.

An “effective amount” of, e.g., a hedgehog agonist, with respect to thesubject method of treatment, refers to an amount of the agonist in apreparation which, when applied as part of a desired dosage regimenbrings about, e.g., a change in the rate of cell proliferation and/orthe state of differentiation of a cell and/or rate of survival of a cellaccording to clinically acceptable standards for the disorder to betreated or the cosmetic purpose.

The terms “epithelia”, “epithelial” and “epithelium” refer to thecellular covering of internal and external body surfaces (cutaneous,mucous and serous), including the glands and other structures derivedtherefrom, e.g., corneal, esophegeal, epidermal, and hair follicleepithelial cells. Other exemplary epithlelial tissue includes: olfactoryepithelium, which is the pseudostratified epithelium lining theolfactory region of the nasal cavity, and containing the receptors forthe sense of smell; glandular epithelium, which refers to epitheliumcomposed of secreting cells; squamous epithelium, which refers toepithelium composed of flattened plate-like cells. The term epitheliumcan also refer to transitional epithelium, like that which ischaracteristically found lining hollow organs that are subject to greatmechanical change due to contraction and distention, e.g., tissue whichrepresents a transition between stratified squamous and columnarepithelium.

The term “epithelialization” refers to healing by the growth ofepithelial tissue over a denuded surface.

The term “epidermal gland” refers to an aggregation of cells associatedwith the epidermis and specialized to secrete or excrete materials notrelated to their ordinary metabolic needs. For example, “sebaceousglands” are holocrine glands in the corium that secrete an oilysubstance and sebum. The term “sweat glands” refers to glands thatsecrete sweat, situated in the corium or subcutaneous tissue, opening bya duct on the body surface.

The term “epidermis” refers to the outermost and nonvascular layer ofthe skin, derived from the embryonic ectoderm, varying in thickness from0.07-1.4 mm. On the palmar and plantar surfaces it comprises, fromwithin outward, five layers: basal layer composed of columnar cellsarranged perpendicularly; prickle-cell or spinous layer composed offlattened polyhedral cells with short processes or spines; granularlayer composed of flattened granular cells; clear layer composed ofseveral layers of clear, transparent cells in which the nuclei areindistinct or absent; and horny layer composed of flattened, cornifiednon-nucleated cells. In the epidermis of the general body surface, theclear layer is usually absent.

“Excisional wounds” include tears, abrasions, cuts, punctures orlacerations in the epithelial layer of the skin and may extend into thedermal layer and even into subcutaneous fat and beyond. Excisionalwounds can result from surgical procedures or from accidentalpenetration of the skin.

The “growth state” of a cell refers to the rate of proliferation of thecell and/or the state of differentiation of the cell. An “altered growthstate” is a growth state characterized by an abnormal rate ofproliferation, e.g., a cell exhibiting an increased or decreased rate ofproliferation relative to a normal cell.

The term “hair” refers to a threadlike structure, especially thespecialized epidermal structure composed of keratin and developing froma papilla sunk in the corium, produced only by mammals andcharacteristic of that group of animals. Also, “hair” may refer to theaggregate of such hairs. A “hair follicle” refers to one of thetubular-invaginations of the epidermis enclosing the hairs, and fromwhich the hairs grow. “Hair follicle epithelial cells” refers toepithelial cells which surround the dermal papilla in the hair follicle,e.g., stem cells, outer root sheath cells, matrix cells, and inner rootsheath cells. Such cells may be normal non-malignant cells, ortransformed/immortalized cells.

The term “hedgehog agonist” refers to an agent which potentiates orrecapitulates the bioactivity of hedgehog, such as to activatetranscription of target genes. Preferred hedgehog agonists can be usedto mimic or enhance the activity or effect of hedgehog protein in asmoothened-dependent manner. The term ‘hedgehog agonist’ as used hereinrefers not only to any agent that may act by directly activating thenormal function of the hedgehog protein, but also to any agent thatactivates the hedgehog signalling pathway, and thus inhibits thefunction of ptc.

The term “hedgehog loss-of-function” refers to an aberrant modificationor mutation of a ptc gene, hedgehog gene, or smoothened gene, or adecrease (or loss) in the level of expression of such a gene, whichresults in a phenotype which resembles contacting a cell with a hedgehoginhibitor, e.g., aberrant inhibition of a hedgehog pathway. Theloss-of-function may include an increase in the ability of the ptc geneproduct to regulate the level of expression of Ci genes, e.g., Gli1,Gli2, and Gli3. The term ‘hedgehog loss-of-function’ is also used hereinto refer to any similar cellular phenotype (e.g., exhibiting reducedproliferation) which occurs due to an alteration anywhere in thehedgehog signal transduction pathway, including, but not limited to, amodification or mutation of hedgehog itself. For example, a cell with anabnormally low proliferation rate due to inactivation of the hedgehogsignalling pathway would have a ‘hedgehog loss-of-function’ phenotype,even if hedgehog is not mutated in that cell.

As used herein, “immortalized cells” refers to cells which have beenaltered via chemical and/or recombinant means such that the cells havethe ability to grow through an indefinite number of divisions inculture.

“Internal epithelial tissue” refers to tissue inside the body which hascharacteristics similar to the epidermal layer in the skin. Examplesinclude the lining of the intestine. The method of the present inventionis useful for promoting the healing of certain internal wounds, forexample wounds resulting from surgery.

The term “keratosis” refers to proliferative skin disorder characterizedby hyperplasia of the horny layer of the epidermis. Exemplary keratoticdisorders include keratosis follicularis, keratosis palmaris etplantaris, keratosis pharyngea, keratosis pilaris, and actinickeratosis.

The term “LD₅₀” means the dose of a drug which is lethal in 50% of testsubjects.

The term “nail” refers to the horny cutaneous plate on the dorsalsurface of the distal end of a finger or toe.

The term “patched gain-of-function” refers to an aberrant modificationor mutation of a ptc gene, or an increased level of expression of thegene, which results in a phenotype which resembles contacting a cellwith a hedgehog inhibitor, e.g., aberrant deactivation of a hedgehogpathway. The gain-of-function may include an increase of the ability ofthe ptc gene product to regulate the level of expression of Ci genes,e.g., Gli1, Gli2 and Gli3.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human animal.

The term “prodrug” is intended to encompass compounds which, underphysiological conditions, are converted into the therapeutically activeagents of the present invention. A common method for making a prodrug isto include selected moieties which are hydrolyzed under physiologicalconditions to reveal the desired molecule. In other embodiments, theprodrug is converted by an enzymatic activity of the host animal.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

Throughout this application, the term “proliferative skin disorder”refers to any disease/disorder of the skin marked by unwanted oraberrant proliferation of cutaneous tissue. These conditions aretypically characterized by epidermal cell proliferation or incompletecell differentiation, and include, for example, X-linked ichthyosis,psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytichyperkeratosis, and seborrheic dermatitis. For example,epidermodysplasia is a form of faulty development of the epidermis.Another example is “epidermolysis”, which refers to a loosened state ofthe epidermis with formation of blebs and bullae either spontaneously orat the site of trauma.

The term “skin” refers to the outer protective covering of the body,consisting of the corium and the epidermis, and is understood to includesweat and sebaceous glands, as well as hair follicle structures.Throughout the present application, the adjective “cutaneous” may beused, and should be understood to refer generally to attributes of theskin, as appropriate to the context in which they are used.

The term “small molecule” refers to a compound having a molecular weightless than about 2500 amu, preferably less than about 2000 amu, even morepreferably less than about 1500 amu, still more preferably less thanabout 1000 amu, or most preferably less than about 750 amu.

The term “smoothened loss-of-function” refers to an aberrantmodification or mutation of a smo gene, or a decreased level ofexpression of the gene, which results in a phenotype which resemblescontacting a cell with a hedgehog inhibitor, e.g., aberrant deactivationof a hedgehog pathway. While not wishing to be bound by any particulartheory, it is noted that ptc may not signal directly into the cell, butrather interact with smoothened, another membrane-bound protein locateddownstream of ptc in hedgehog signaling (Marigo et al., (1996) Nature384: 177-179). The gene smo is a segment-polarity gene required for thecorrect patterning of every segment in Drosophila (Alcedo et al., (1996)Cell 86: 221-232). Human homologs of smo have been identified. See, forexample, Stone et al. (1996) Nature 384:129-134, and GenBank accessionU84401. The smoothened gene encodes an integral membrane protein withcharacteristics of heterotrimeric G-protein-coupled receptors; i.e.,7-transmembrane regions. This protein shows homology to the DrosophilaFrizzled (Fz) protein, a member of the wingless pathway. It wasoriginally thought that smo encodes a receptor of the Hh signal.However, this suggestion was subsequently disproved, as evidence for ptcbeing the Hh receptor was obtained. Cells that express Smo fail to bindHh, indicating that smo does not interact directly with Hh (Nusse,(1996) Nature 384: 119-120). Rather, the binding of Sonic hedgehog (SHH)to its receptor, PTCH, is thought to prevent normal inhibition by PTCHof smoothened (SMO), a seven-span transmembrane protein.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above.

Herein, the term “aliphatic group” refers to a straight-chain,branched-chain, or cyclic aliphatic hydrocarbon group and includessaturated and unsaturated aliphatic groups, such as an alkyl group, analkenyl group, and an alkynyl group.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), and more preferably 20 or fewer. Likewise, preferredcycloalkyls have from 3-10 carbon atoms in their ring structure, andmore preferably have 5, 6 or 7 carbons in the ring structure.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, anamido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl,an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethylthio, and thelike.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈, or R₉ and R₁₀ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In preferred embodiments, only one of R₉ or R₁₀can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not forman imide. In still more preferred embodiments, the term ‘amine’ does notencompass amides, e.g., wherein one of R₉ and R₁₀ represents a carbonyl.In even more preferred embodiments, R₉ and R₁₀ (and optionally R′₁₀)each independently represent a hydrogen, an alkyl, an alkenyl, or—(CH₂)_(m)—R₈. Thus, the term “alkylamine” as used herein means an aminegroup, as defined above, having a substituted or unsubstituted alkylattached thereto, i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thioformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, anaromatic or heteroaromatic moiety, —CF₃, —CN, or the like.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

A “phosphonamidite” can be represented in general formula:

wherein R₉ and R₁₀ are as defined above, Q₂ represents O, S or N, andR₄₈ represents a lower alkyl or an aryl, Q₂ represents O, S or N.

A “phosphoramidite” can be represented in general formula:

wherein R₉ and R₁₀ are as defined above, and Q₂ represents O, S or N.

A “phosphoryl” can in general be represented by the formula:

wherein Q₁ represented S or O, and R₄₆ represents hydrogen, a loweralkyl or an aryl. When used to substitute, for example, an alkyl, thephosphoryl group of the phosphorylalkyl can be represented by thegeneral formula:

wherein Q₁ represented S or O, and each R₄₆ independently representshydrogen, a lower alkyl or an aryl, Q₂ represents O, S or N. When Q₁ isan S, the phosphoryl moiety is a “phosphorothioate”.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R_(8,) m and R₈ beingdefined above.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₉ and R₁₀ are as defined above.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that canbe represented by the general formula:

in which R₉ and R′₁₁ are as defined above.

The term “sulfonate” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts may be formed with an appropriateoptically active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., the ability to activate hedgehogsignaling), wherein one or more simple variations of substituents aremade which do not adversely affect the efficacy of the compound. Ingeneral, the compounds of the present invention may be prepared by themethods illustrated in general reaction schemes as, for example,described below, or by modifications thereof, using readily availablestarting materials, reagents and conventional synthesis procedures. Inthese reactions, it is also possible to make use of variants which arein themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and non-aromatic organic compounds which can besubstituted or unsubstituted.

III. Exemplary Compounds of the Invention

As described in further detail below, it is contemplated that thesubject methods can be carried out using a variety of different smallmolecules which can be readily identified, for example, by such drugscreening assays as described herein. For example, compounds useful inthe subject methods include compounds represented by general formula(I):

wherein, as valence and stability permit,

Ar and Ar′ independently represent substituted or unsubstituted aryl orheteroaryl rings;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

Cy and Cy′ independenly represent substituted or unsubstituted aryl,heterocyclyl, heteroaryl, or cycloalkyl, including polycyclic groups;and

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Ar and Ar′ represent phenyl rings, e.g.,unsubstituted or substituted with one or more groups includingheteroatoms such as O, N, and S. In certain embodiments, at least one ofAr and Ar′ represents a phenyl ring. In certain embodiments, at leastone of Ar and Ar′ represents a heteroaryl ring, e.g., a pyridyl,thiazolyl, thienyl, pyrimidyl, etc. In certain embodiments, Y and Ar′are attached to Ar in a meta and/or 1,3-relationship.

In certain embodiments, Y is absent from all positions. In embodimentswherein Y is present in a position, i preferably represents an integerfrom 1-2 in an adjacent M_(i) if i=0 would result in two occurrences ofY being directly attached, or an occurrence of Y being directly attachedto N.

In certain embodiments, Cy′ is a substituted or unsubstituted aryl orheteroaryl. In certain embodiments, Cy′ is directly attached to X. Incertain embodiments, Cy′ is a substituted or unsubstituted bicyclic orheteroaryl ring, preferably both bicyclic and heteroaryl, such asbenzothiophene, benzofuran, benzopyrrole, benzopyridine, etc. In certainembodiments, Cy′ is a monocyclic aryl or heteroaryl ring substituted atleast with a substituted or unsubstituted aryl or heteroaryl ring, i.e.,forming a biaryl system. In certain embodiments, Cy′ includes twosubstituted or unsubstituted aryl or heteroaryl rings, e.g., the same ordifferent, directly connected by one or more bonds, e.g., to form abiaryl or bicyclic ring system. In certain embodiments, Cy′ represents abenzo(b)thien-2-yl, preferably a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl, e.g.,wherein the benzo ring is substituted with from 1-4 substituentsselected from halogen, nitro, cyano, methyl (e.g., including halomethyl,such as CHCl₂ and CF₃), and ethyl (e.g., including haloethyl, such asCH₂CCl₃, C₂F₅, etc.), preferably from halogen and methyl (e.g.,including halomethyl, such as CHCl₂ and CF₃). In certain suchembodiments Cy′ represents a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl wherein thebenzo ring is substituted with fluoro at the 4-position (peri to the3-substituent on the thienyl ring) and, optionally, at the 7-position(‘peri’ to the S of the thienyl ring).

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, R represents H or lower alkyl, e.g., H or Me.

In certain embodiments, Cy represents a substituted or unsubstitutednon-aromatic carbocyclic or heterocyclic ring, i.e., including at leastone sp³ hybridized atom, and preferably a plurality of sp³ hybridizedatoms. In certain embodiments, Cy includes an amine within the atoms ofthe ring or on a substituent of the ring, e.g., Cy is pyridyl,imidazolyl, pyrrolyl, piperidyl, pyrrolidyl, piperazyl, etc., and/orbears an amino substituent. In certain embodiments, Cy is a 5- to7-membered ring. In certain embodiments, Cy is directly attached to N.In embodiments wherein Cy is a six-membered ring directly attached to Nand bears an amino substituent at the 4 position of the ring relative toN, the N and amine substituents may be disposed trans on the ring.

In certain embodiments, substituents on Ar or Ar′ are selected fromhalogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove, wherein p and n, individually for each occurrence, representintegers from 0 to 10, preferably from 0 to 5.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (II):

wherein, as valence and stability permit,

Ar and Ar′ independently represent substituted or unsubstituted aryl orheteroaryl rings;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—;

X is selected from —C(═O)—, —C(═S)—, —S(O2)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH2—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne, wherein some or all occurrences of M inM_(j) form all or part of a cyclic structure;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

Cy′ represents a substituted or unsubstituted aryl, heterocyclyl,heteroaryl, or cycloalkyl, including polycyclic groups;

j represents, independently for each occurrence, an integer from 0 to10, preferably from 2 to 7; and

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Ar and Ar′ represent phenyl rings, e.g.,unsubstituted or substituted with one or more groups includingheteroatoms such as O, N, and S. In certain embodiments, at least one ofAr and Ar′ represents a phenyl ring. In certain embodiments, at leastone of Ar and Ar′ represents a heteroaryl ring, e.g., a pyridyl,thiazolyl, thienyl, pyrimidyl, etc. In certain embodiments, Y and Ar′are attached to Ar in a meta and/or 1,3-relationship.

In certain embodiments, Y is absent from all positions. In embodimentswherein Y is present in a position, i preferably represents an integerfrom 1-2 in an adjacent M_(i) if i=0 would result in two occurrences ofY being directly attached, or an occurrence of Y being directly attachedto N or NR₂.

In certain embodiments, Cy′ is a substituted or unsubstituted aryl orheteroaryl. In certain embodiments, Cy′ is directly attached to X. Incertain embodiments, Cy′ is a substituted or unsubstituted bicyclic orheteroaryl ring, preferably both bicyclic and heteroaryl, such asbenzothiophene, benzofuran, benzopyrrole, benzopyridine, etc. In certainembodiments, Cy′ is a monocyclic aryl or heteroaryl ring substituted atleast with a substituted or unsubstituted aryl or heteroaryl ring, i.e.,forming a biaryl system. In certain embodiments, Cy′ includes twosubstituted or unsubstituted aryl or heteroaryl rings, e.g., the same ordifferent, directly connected by one or more bonds, e.g., to form abiaryl or bicyclic ring system. In certain embodiments, Cy′ represents abenzo(b)thien-2-yl, preferably a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl, e.g.,wherein the benzo ring is substituted with from 1-4 substituentsselected from halogen, nitro, cyano, methyl (e.g., including halomethyl,such as CHCl₂ and CF₃), and ethyl (e.g., including haloethyl, such asCH₂CCl₃, C₂F₅, etc.), preferably from halogen and methyl (e.g.,including halomethyl, such as CHCl₂ and CF₃). In certain suchembodiments Cy′ represents a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl wherein thebenzo ring is substituted with fluoro at the 4-position (peri to the3-substituent on the thienyl ring) and, optionally, at the 7-position(‘peri’ to the S of the thienyl ring).

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, R represents H or lower alkyl, e.g., H or Me.

In certain embodiments, NR₂ represents a primary amine or a secondary ortertiary amine substituted with one or two lower alkyl groups, arylgroups, or aralkyl groups, respectively, preferably a primary amine orsecondary amine.

In certain embodiments, substituents on Ar or Ar′ are selected fromhalogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove, wherein p and n, individually for each occurrence, representintegers from 0 to 10, preferably from 0 to 5.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (III):

wherein, as valence and stability permit,

Ar and Ar′ independently represent substituted or unsubstituted aryl orheteroaryl rings;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

Cy and Cy′ independenly represent substituted or unsubstituted aryl,heterocyclyl, heteroaryl, or cycloalkyl, including polycyclic groups;and

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Ar and Ar′ represent phenyl rings, e.g.,unsubstituted or substituted with one or more groups includingheteroatoms such as O, N, and S. In certain embodiments, at least one ofAr and Ar′ represents a phenyl ring. In certain embodiments, at leastone of Ar and Ar′ represents a heteroaryl ring, e.g., a pyridyl,thiazolyl, thienyl, pyrimidyl, etc. In certain embodiments, Y and Ar′are attached to Ar in a meta and/or 1,3-relationship.

In certain embodiments, Y is absent from all positions. In embodimentswherein Y is present in a position, i preferably represents an integerfrom 1-2 in an adjacent M_(i) if i=0 would result in two occurrences ofY being directly attached, or an occurrence of Y being directly attachedto N or NR₂.

In certain embodiments, Cy′ is a substituted or unsubstituted aryl orheteroaryl. In certain embodiments, Cy′ is directly attached to X. Incertain embodiments, Cy′ is a substituted or unsubstituted bicyclic orheteroaryl ring, preferably both bicyclic and heteroaryl, such asbenzothiophene, benzofuran, benzopyrrole, benzopyridine, etc. In certainembodiments, Cy′ is a monocyclic aryl or heteroaryl ring substituted atleast with a substituted or unsubstituted aryl or heteroaryl ring, i.e.,forming a biaryl system. In certain embodiments, Cy′ includes twosubstituted or unsubstituted aryl or heteroaryl rings, e.g., the same ordifferent, directly connected by one or more bonds, e.g., to form abiaryl or bicyclic ring system. In certain embodiments, Cy′ represents abenzo(b)thien-2-yl, preferably a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl, e.g.,wherein the benzo ring is substituted with from 1-4 substituentsselected from halogen, nitro, cyano, methyl (e.g., including halomethyl,such as CHCl₂ and CF₃), and ethyl (e.g., including haloethyl, such asCH₂CCl₃, C₂F₅, etc.), preferably from halogen and methyl (e.g.,including halomethyl, such as CHCl₂ and CF₃). In certain suchembodiments Cy′ represents a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl wherein thebenzo ring is substituted with fluoro at the 4-position (peri to the3-substituent on the thienyl ring) and, optionally, at the 7-position(‘peri’ to the S of the thienyl ring).

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, R represents H or lower alkyl, e.g., H or Me.

In certain embodiments, NR₂ represents a primary amine or a secondary ortertiary amine substituted with one or two lower alkyl groups, arylgroups, or aralkyl groups, respectively, preferably a primary amine or asecondary amine.

In certain embodiments, Cy represents a substituted or unsubstitutednon-aromatic carbocyclic or heterocyclic ring, i.e., including at leastone sp³ hybridized atom, and preferably a plurality of sp³ hybridizedatoms. In certain embodiments, Cy is directly attached to N and/or toNR₂. In certain embodiments, Cy is a 5- to 7-membered ring. Inembodiments wherein Cy is a six-membered ring directly attached to N andbears an amino substituent at the 4 position of the ring relative to N,the N and amine substituents may be disposed trans on the ring.

In certain embodiments, substituents on Ar or Ar′ are selected fromhalogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove, wherein n and p, individually for each occurrence, representintegers from 0 to 10, preferably from 0 to 5.

In certain embodiments, compounds useful in the subject methods includecompounds represented by general formula (IV):

wherein, as valence and stability permit,

Cy′ represents a substituted or unsubstituted aryl or heteroaryl ring,including polycyclics;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

R₁ and R₂ represent, independently and as valency permits, from 0-5substituents on the ring to which it is attached, selected from halogen,lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl,ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove;

Cy represents substituted or unsubstituted aryl, heterocyclyl,heteroaryl, or cycloalkyl, including polycyclic groups;

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2; and

p and n, individually for each occurrence, represent integers from 0 to10, preferably from 0 to 5.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Cy′ represents a substituted or unsubstitutedbicyclic or heterocyclic ring system, preferably both bicyclic andheteroaryl, such as benzothiophene, benzofuran, benzopyrrole,benzopyridine, etc. In certain embodiments, Cy′ is directly attached toX. In certain embodiments, Cy′ is a monocyclic aryl or heteroaryl ringsubstituted at least with a substituted or unsubstituted aryl orheteroaryl ring, i.e., forming a biaryl system. In certain embodiments,Cy′ includes two substituted or unsubstituted aryl or heteroaryl rings,e.g., the same or different, directly connected by one or more bonds,e.g., to form a biaryl or bicyclic ring system. In certain embodiments,Cy′ represents a benzo(b)thien-2-yl, preferably a3-chloro-benzo(b)thien-2-yl, 3-fluoro-benzo(b)thien-2-yl, or3-methyl-benzo(b)thien-2-yl, e.g., wherein the benzo ring is substitutedwith from 1-4 substituents selected from halogen, nitro, cyano, methyl(e.g., including halomethyl, such as CHCl₂ and CF₃), and ethyl (e.g.,including haloethyl, such as CH₂CCl₃, C₂F₅, etc.), preferably fromhalogen and methyl (e.g., including halomethyl, such as CHCl₂ and CF₃).In certain such embodiments Cy′ represents a3-chloro-benzo(b)thien-2-yl, 3-fluoro-benzo(b)thien-2-yl, or3-methyl-benzo(b)thien-2-yl wherein the benzo ring is substituted withfluoro at the 4-position (peri to the 3-substituent on the thienyl ring)and, optionally, at the 7-position (‘peri’ to the S of the thienylring).

In certain embodiments, Y is absent from all positions. In embodimentswherein Y is present in a position, i preferably represents an integerfrom 1-2 in an adjacent M_(i) if i=0 would result in two occurrences ofY being directly attached, or an occurrence of Y being directly attachedto N.

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, R represents H or lower alkyl, e.g., H or Me.

In certain embodiments, Cy represents a substituted or unsubstitutednon-aromatic carbocyclic or heterocyclic ring, i.e., including at leastone sp³ hybridized atom, and preferably a plurality of sp³ hybridizedatoms. In certain embodiments, Cy is directly attached to N. In certainembodiments, Cy is a 5- to 7-membered ring. In embodiments wherein Cy isa six-membered ring directly attached to N and bears an aminosubstituent at the 4 position of the ring relative to N, the N and aminesubstituents may be disposed trans on the ring.

In certain embodiments, R₁ and R₂ represent, independently and asvalency permits, from 0-5 substituents on the ring to which it isattached, selected from halogen, lower alkyl, lower alkenyl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, —(CH₂)_(p)alkyl, —(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (V):

wherein, as valence and stability permit,

Cy′ represents a substituted or unsubstituted aryl or heteroaryl ring,including polycyclics;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

R₁ and R₂ represent, independently and as valency permits, from 0-5substituents on the ring to which it is attached, selected from halogen,lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl,ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove;

Cy′ represents a substituted or unsubstituted aryl, heterocyclyl,heteroaryl, or cycloalkyl, including polycyclic groups;

j represents, independently for each occurrence, an integer from 0 to10, preferably from 2 to 7;

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2; and

p and n, individually for each occurrence, represent integers from 0 to10, preferably from 0 to 5.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Cy′ represents a substituted or unsubstitutedbicyclic or heterocyclic ring system, preferably both bicyclic andheteroaryl, such as benzothiophene, benzofuran, benzopyrrole,benzopyridine, etc. In certain embodiments, Cy′ is directly attached toX. In certain embodiments, Cy′ is a monocyclic aryl or heteroaryl ringsubstituted at least with a substituted or unsubstituted aryl orheteroaryl ring, i.e., forming a biaryl system. In certain embodiments,Cy′ includes two substituted or unsubstituted aryl or heteroaryl rings,e.g., the same or different, directly connected by one or more bonds,e.g., to form a biaryl or bicyclic ring system. In certain embodiments,Cy′ represents a benzo(b)thien-2-yl, preferably a3-chloro-benzo(b)thien-2-yl, 3-fluoro-benzo(b)thien-2-yl, or3-methyl-benzo(b)thien-2-yl, e.g., wherein the benzo ring is substitutedwith from 1-4 substituents selected from halogen, nitro, cyano, methyl(e.g., including halomethyl, such as CHCl₂ and CF₃), and ethyl (e.g.,including haloethyl, such as CH₂CCl₃, C₂F₅, etc.), preferably fromhalogen and methyl (e.g., including halomethyl, such as CHCl₂ and CF₃).In certain such embodiments Cy′ represents a3-chloro-benzo(b)thien-2-yl, 3-fluoro-benzo(b)thien-2-yl, or3-methyl-benzo(b)thien-2-yl wherein the benzo ring is substituted withfluoro at the 4-position (peri to the 3-substituent on the thienyl ring)and, optionally, at the 7-position (‘peri’ to the S of the thienylring).

In certain embodiments, Y is absent from all positions. In embodimentswherein Y is present in a position, i preferably represents an integerfrom 1-2 in an adjacent M_(i) if i=0 would result in two occurrences ofY being directly attached, or an occurrence of Y being directly attachedto N or NR₂.

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, NR₂ represents a primary amine or a secondary ortertiary amine substituted with one or two lower alkyl groups, arylgroups, or aralkyl groups, respectively, preferably a primary orsecondary amine.

In certain embodiments, R represents H or lower alkyl, e.g., H or Me.

In certain embodiments, R₁ and R₂ represent, independently and asvalency permits, from 0-5 substituents on the ring to which it isattached, selected from halogen, lower alkyl, lower alkenyl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, —(CH₂)_(p)alkyl, —(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (VI):

Formula VI

wherein, as valence and stability permit,

Cy′ represents a substituted or unsubstituted aryl or heteroaryl ring,including polycyclics;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

Cy represents substituted or unsubstituted aryl, heterocyclyl,heteroaryl, or cycloalkyl, including polycyclic groups;

R₁ and R₂ represent, independently and as valency permits, from 0-5substituents on the ring to which it is attached, selected from halogen,lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl,ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove;

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2; and

n and p, individually for each occurrence, represent integers from 0 to10, preferably from 0 to 5.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Cy′ represents a substituted or unsubstitutedbicyclic or heteroaryl ring system, preferably both bicyclic andheteroaryl, e.g., benzothiophene, benzofuran, benzopyrrole,benzopyridyl, etc. In certain embodiments, Cy′ is directly attached toX. In certain embodiments, Cy′ is a monocyclic aryl or heteroaryl ringsubstituted at least with a substituted or unsubstituted aryl orheteroaryl ring, i.e., forming a biaryl system. In certain embodiments,Cy′ includes two substituted or unsubstituted aryl or heteroaryl rings,e.g., the same or different, directly connected by one or more bonds,e.g., to form a biaryl or bicyclic ring system. In certain embodiments,Cy′ represents a benzo(b)thien-2-yl, preferably a3-chloro-benzo(b)thien-2-yl, 3-fluoro-benzo(b)thien-2-yl, or3-methyl-benzo(b)thien-2-yl, e.g., wherein the benzo ring is substitutedwith from 1-4 substituents selected from halogen, nitro, cyano, methyl(e.g., including halomethyl, such as CHCl₂ and CF₃), and ethyl (e.g.,including haloethyl, such as CH₂CCl₃, C₂F₅, etc.), preferably fromhalogen and methyl (e.g., including halomethyl, such as CHCl₂ and CF₃).In certain such embodiments Cy′ represents a3-chloro-benzo(b)thien-2-yl, 3-fluoro-benzo(b)thien-2-yl, or3-methyl-benzo(b)thien-2-yl wherein the benzo ring is substituted withfluoro at the 4-position (peri to the 3-substituent on the thienyl ring)and, optionally, at the 7-position (‘peri’ to the S of the thienylring).

In certain embodiments, Y is absent from all positions. In embodimentswherein Y is present in a position, i preferably represents an integerfrom 1-2 in an adjacent M_(i) if i=0 would result in two occurrences ofY being directly attached, or an occurrence of Y being directly attachedto N or NR₂.

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, NR₂ represents a primary amine or a secondary ortertiary amine substituted with one or two lower alkyl groups, arylgroups, or aralkyl groups, respectively, preferably a primary amine.

In certain embodiments, R represents H or lower alkyl, e.g., H or Me.

In certain embodiments, Cy represents a substituted or unsubstitutednon-aromatic carbocyclic or heterocyclic ring, i.e., including at leastone sp³ hybridized atom, and preferably a plurality of sp³ hybridizedatoms. In certain embodiments, Cy is directly attached to N and/or toNR₂. In certain embodiments, Cy is a 5- to 7-membered ring. Inembodiments wherein Cy is a six-membered ring directly attached to N andbears an amino substituent at the 4 position of the ring relative to N,the N and amine substituents may be disposed trans on the ring.

In certain embodiments, R₁ and R₂ represent, independently and asvalency permits, from 0-5 substituents on the ring to which it isattached, selected from halogen, lower alkyl, lower alkenyl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, —(CH₂)_(p)alkyl, —(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove.

In certain embodiments, a subject compound has the structure of FormulaVII:

wherein, as valence and stability permit,

Cy represents a substituted or unsubstituted heterocyclyl or cycloalkyl;

Cy′ is a substituted or unsubstituted aryl or heteroaryl ring, includingpolycyclics;

W is O or S;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl,alkynyl, alkenyl, or alkyl, or two R taken together may form a 4- to8-membered ring, e.g., with N;

R₁ and R₂ represent, independently and as valency permits, from 0-5substituents on the ring to which it is attached, selected from halogen,lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl, thiocarbonyl,ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro,hydroxyl, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, —(CH₂)_(p)alkyl,—(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl, —(CH₂)_(p)aralkyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove;

n and p, individually for each occurrence, represent integers from 0 to10, preferably from 0 to 5.

In certain embodiments, Cy′ represents a substituted or unsubstitutedbicyclic or heteroaryl ring system, preferably both bicyclic andheteroaryl, e.g., benzothiophene, benzofuran, benzopyrrole,benzopyridyl, etc. In certain other embodiments, Cy′ represents an arylor heteroaryl ring substituted at least with a substituted orunsubstituted aryl or heteroaryl ring, i.e., to form a biaryl ringsystem. In certain embodiments, Cy′ represents a benzo(b)thien-2-yl,preferably a 3-chloro-benzo(b)thien-2-yl, 3-fluoro-benzo(b)thien-2-yl,or 3-methyl-benzo(b)thien-2-yl, e.g., wherein the benzo ring issubstituted with from 1-4 substituents selected from halogen, nitro,cyano, methyl (e.g., including halomethyl, such as CHCl₂ and CF₃), andethyl (e.g., including haloethyl, such as CH₂CCl₃, C₂F₅, etc.),preferably from halogen and methyl (e.g., including halomethyl, such asCHCl₂ and CF₃). In certain such embodiments Cy′ represents a3-chloro-benzo(b)thien-2-yl, 3-fluoro-benzo(b)thien-2-yl, or3-methyl-benzo(b)thien-2-yl wherein the benzo ring is substituted withfluoro at the 4-position (peri to the 3-substituent on the thienyl ring)and, optionally, at the 7-position (‘peri’ to the S of the thienylring).

In certain embodiments, NR₂ represents a primary amine or a secondary ortertiary amine substituted with one or two lower alkyl groups, arylgroups, or aralkyl groups, respectively, preferably a primary orsecondary amine.

In certain embodiments, Cy represents a substituted or unsubstitutedsaturated carbocyclic or heterocyclic ring, i.e., composed of aplurality of sp³ hybridized atoms. In certain embodiments, Cy is a 5- to7-membered ring. In embodiments wherein Cy is a six-membered ringdirectly attached to N and bears an amino substituent at the 4 positionof the ring relative to N, the N and amine substituents may be disposedtrans on the ring.

In certain embodiments, R₁ and R₂ represent, independently and asvalency permits, from 0-5 substituents on the ring to which it isattached, selected from halogen, lower alkyl, lower alkenyl, carbonyl,thiocarbonyl, ketone, aldehyde, amino, acylamino, cyano, nitro,hydroxyl, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, —(CH₂)_(p)alkyl, —(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl,—(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl, —(CH₂)_(p)O-lower alkenyl,—O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-lower alkyl, —(CH₂)_(p)S-loweralkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂, —(CH₂)_(p)NR-lower alkyl,—(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, and protected forms of theabove.

In certain embodiments, a subject compound has a structure of FormulaVIII:

wherein, as valence and stability permit,

U represents a substituted or unsubstituted aryl or heteroaryl ringfused to the nitrogen-containing ring;

V represents a lower alkylene group, such as methylene, 1,2-ethylene,1,1-ethylene, 1,1-propylene, 1,2-propylene, 1,3-propylene, etc.;

W represents S or O, preferably O;

X represents C═O, C═S, or SO₂;

R₃ represents substituted or unsubstituted aryl, heteroaryl, loweralkyl, lower alkenyl, lower alkynyl, carbocyclyl, carbocyclylalkyl,heterocyclyl, heterocyclylalkyl, aralkyl, or heteroaralkyl;

R₄ represents substituted or unsubstituted aralkyl or lower alkyl, suchas phenethyl, benzyl, or aminoalkyl, etc.;

R₅ represents substituted or unsubstituted aryl, heteroaryl, aralkyl, orheteroaralkyl, including polycyclic aromatic or heteroaromatic groups.

In certain embodiments, U represents a phenyl ring fused to thenitrogen-containing ring.

In certain embodiments, R₃ is selected from substituted or unsubstitutedaryl, heteroaryl, lower alkyl, lower alkenyl, aralkyl, andheteroaralkyl.

In certain embodiments, R₄ is an unsubstituted lower alkyl group, or isa lower alkyl group substituted with a secondary or tertiary amine.

In certain embodiments, R₅ is selected from substituted or unsubstitutedphenyl or naphthyl, or is a diarylalkyl group, such as2,2-diphenylethyl, diphenylmethyl, etc.

In certain embodiments, subject compounds include compounds representedby general formula (IX):

Formula IX

wherein, as valence and stability permit,

Ar represents a substituted or unsubstituted aryl or heteroaryl ring;

Z is absent or represents a substituted or unsubstituted aryl,carbocyclyl, heterocyclyl, or heteroaryl ring, or a lower alkyl, nitro,cyano, or halogen substituent;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—, provided that if Z is not a ring, then Y attached toZ is absent;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, carbocyclyl, heteroaryl, aralkyl,heteroaralkyl, heterocyclylalkyl, carbocyclylalkyl, alkynyl, alkenyl, oralkyl, or two R taken together may form a 4- to 8-membered ring, e.g.,with N;

Cy and Cy′ independently represent substituted or unsubstituted aryl,heterocyclyl, heteroaryl, or cycloalkyl, including polycyclic groups;

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2; and

k represents an integer from 0 to 3, preferably from 0 to 2.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc. In certain embodiments, i represents 0for all occurrences except in the sequence N—M_(i)—Y—Ar, where irepresents 1.

In certain embodiments, Ar and X independently represent substituted orunsubstituted aryl or heteroaryl rings, e.g., unsubstituted orsubstituted with one or more groups optionally including heteroatomssuch as O, N, and S. In certain embodiments, Ar represents a phenylring. In certain embodiments, at least one of Ar represents a heteroarylring, e.g., a pyridyl, thiazolyl, thienyl, pyrimidyl, furanyl, etc. Incertain embodiments, the occurrences of Y attached to Ar are disposed ina meta and/or 1,3-relationship.

In certain embodiments, Y is absent from all positions. In certainembodiments, the only present occurrence of Y is attached to M_(k). Inembodiments wherein Y is present in a position, i or k preferablyrepresents 2 in an adjacent M_(i/k) if i/k=0 would result in twooccurrences of Y being directly attached to each other, or an occurrenceof Y being directly attached to N. In certain embodiments, where twooccurrences of Y are attached to M, at least one such occurrence of Y isabsent. In certain embodiments, no more than two occurrences of Y arepresent.

In certain embodiments, Cy′ is a substituted or unsubstituted aryl orheteroaryl. In certain embodiments, Cy′ is directly attached to X. Incertain embodiments, Cy′ is a substituted or unsubstituted bicyclic orheteroaryl ring, preferably both bicyclic and heteroaryl, such asbenzothiophene, benzofuran, benzopyrrole, benzopyridine, etc. In certainembodiments, Cy′ is a monocyclic aryl or heteroaryl ring substituted atleast with a substituted or unsubstituted aryl or heteroaryl ring, i.e.,forming a biaryl system. In certain embodiments, Cy′ includes twosubstituted or unsubstituted aryl or heteroaryl rings, e.g., the same ordifferent, directly connected by one or more bonds, e.g., to form abiaryl or bicyclic ring system. In certain embodiments, Cy′ represents abenzo(b)thien-2-yl, preferably a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl, e.g.,wherein the benzo ring is substituted with from 1-4 substituentsselected from halogen, nitro, cyano, methyl (e.g., including halomethyl,such as CHCl₂ and CF₃), and ethyl (e.g., including haloethyl, such asCH₂CCl₃, C₂F₅, etc.), preferably from halogen and methyl (e.g.,including halomethyl, such as CHCl₂ and CF₃). In certain suchembodiments Cy′ represents a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl wherein thebenzo ring is substituted with fluoro at the 4-position (peri to the3-substituent on the thienyl ring) and, optionally, at the 7-position(‘peri’ to the S of the thienyl ring).

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, Cy represents a substituted or unsubstitutednon-aromatic carbocyclic or heterocyclic ring, i.e., including at leastone sp³ hybridized atom, and preferably a plurality of sp³ hybridizedatoms. In certain embodiments, Cy includes an amine within the atoms ofthe ring or on a substituent of the ring, e.g., Cy is pyridyl,imidazolyl, pyrrolyl, piperidyl, pyrrolidyl, piperazyl, etc., and/orbears an amino substituent. In certain embodiments, Cy is a 5- to7-membered ring. In certain embodiments, Cy is directly attached to N.In embodiments wherein Cy is a six-membered ring directly attached to Nand bears an amino substituent at the 4 position of the ring relative toN, the N and amine substituents may be disposed trans on the ring.

In certain embodiments, substituents on Ar or Z, where Z is an aryl orheteroaryl ring, are selected from halogen, lower alkyl, lower alkenyl,aryl, heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,acylamino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,—(CH₂)_(p)alkyl, —(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl,—(CH₂)_(p)aralkyl, —(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl,—(CH₂)_(p)O-lower alkenyl, —O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-loweralkyl, —(CH₂)_(p)S-lower alkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂,—(CH₂)_(p)NR-lower alkyl, —(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, andprotected forms of the above, wherein n and p, individually for eachoccurrence, represent integers from 0 to 10, preferably from 0 to 5.

In certain embodiments, Z is directly attached to Ar, or attached to Arthrough a chain of one or two atoms. In certain embodiments, Z-Y-M,taken together, is absent.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (X):

Formula X

wherein, as valence and stability permit,

Ar represents a substituted or unsubstituted aryl or heteroaryl ring;

Z is absent or represents a substituted or unsubstituted aryl,carbocyclyl, heterocyclyl, or heteroaryl ring, or a lower alkyl, nitro,cyano, or halogen substituent;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—, provided that if Z is not a ring, then Y attached toZ is absent;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, carbocyclyl, heteroaryl, aralkyl,heteroaralkyl, heterocyclylalkyl, carbocyclylalkyl, alkynyl, alkenyl, oralkyl, or two R taken together may form a 4- to 8-membered ring, e.g.,with N;

Cy′ represents a substituted or unsubstituted aryl, heterocyclyl,heteroaryl, or cycloalkyl, including polycyclic groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne, wherein some or all occurrences of M inM_(j) form all or part of a cyclic structure;

j represents, independently for each occurrence, an integer from 2 to10, preferably from 2 to 7;

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2; and

k represents an integer from 0 to 3, preferably from 0 to 2.

In certain embodiments, NR₂ represents a primary amine or a secondary ortertiary amine substituted with one or two lower alkyl groups, arylgroups, or aralkyl groups, respectively, preferably a primary orsecondary amine.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc. In certain embodiments, i represents 0for all occurrences except in the sequence N—M_(i)—Y—Ar, where irepresents 1.

In certain embodiments, Ar and X independently represent substituted orunsubstituted aryl or heteroaryl rings, e.g., unsubstituted orsubstituted with one or more groups optionally including heteroatomssuch as O, N, and S. In certain embodiments, Ar represents a phenylring. In certain embodiments, Ar represents a heteroaryl ring, e.g., apyridyl, thiazolyl, thienyl, pyrimidyl, furanyl, etc. In certainembodiments, the occurrences of Y attached to Ar are disposed in a metaand/or 1,3-relationship.

In certain embodiments, Y is absent from all positions. In certainembodiments, the only present occurrence of Y is attached to M_(k). Inembodiments wherein Y is present in a position, i or k preferablyrepresents 2 in an adjacent M_(i/k) if i/k=0 would result in twooccurrences of Y being directly attached to each other, or an occurrenceof Y being directly attached to N. In certain embodiments, where twooccurrences of Y are attached to M, at least one such occurrence of Y isabsent. In certain embodiments, no more than two occurrences of Y arepresent.

In certain embodiments, Cy′ is a substituted or unsubstituted aryl orheteroaryl. In certain embodiments, Cy′ is directly attached to X. Incertain embodiments, Cy′ is a substituted or unsubstituted bicyclic orheteroaryl ring, preferably both bicyclic and heteroaryl, such asbenzothiophene, benzofuran, benzopyrrole, benzopyridine, etc. In certainembodiments, Cy′ is a monocyclic aryl or heteroaryl ring substituted atleast with a substituted or unsubstituted aryl or heteroaryl ring, i.e.,forming a biaryl system. In certain embodiments, Cy′ includes twosubstituted or unsubstituted aryl or heteroaryl rings, e.g., the same ordifferent, directly connected by one or more bonds, e.g., to form abiaryl or bicyclic ring system. In certain embodiments, Cy′ represents abenzo(b)thien-2-yl, preferably a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl, e.g.,wherein the benzo ring is substituted with from 1-4 substituentsselected from halogen, nitro, cyano, methyl (e.g., including halomethyl,such as CHCl₂ and CF₃), and ethyl (e.g., including haloethyl, such asCH₂CCl₃, C₂F₅, etc.), preferably from halogen and methyl (e.g.,including halomethyl, such as CHCl₂ and CF₃). In certain suchembodiments Cy′ represents a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl wherein thebenzo ring is substituted with fluoro at the 4-position (peri to the3-substituent on the thienyl ring) and, optionally, at the 7-position(‘peri’ to the S of the thienyl ring).

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, substituents on Ar or Z, where Z is an aryl orheteroaryl ring, are selected from halogen, lower alkyl, lower alkenyl,aryl, heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,acylamino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,—(CH₂)_(p)alkyl, —(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl,—(CH₂)_(p)aralkyl, —(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl,—(CH₂)_(p)O-lower alkenyl, —O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-loweralkyl, —(CH₂)_(p)S-lower alkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂,—(CH₂)_(p)NR-lower alkyl, —(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, andprotected forms of the above, wherein n and p, individually for eachoccurrence, represent integers from 0 to 10, preferably from 0 to 5.

In certain embodiments, Z is directly attached to Ar, or attached to Arthrough a chain of one or two atoms. In certain embodiments, Z-Y-M,taken together, is absent.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (XI):

Formula XI

wherein, as valence and stability permit,

Ar represents a substituted or unsubstituted aryl or heteroaryl ring;

Z is absent or represents a substituted or unsubstituted aryl,carbocyclyl, heterocyclyl, or heteroaryl ring, or a lower alkyl, nitro,cyano, or halogen substituent;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—, provided that if Z is not a ring, then Y attached toZ is absent;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne;

R represents, independently for each occurrence, H or substituted orunsubstituted aryl, heterocyclyl, carbocyclyl, heteroaryl, aralkyl,heteroaralkyl, heterocyclylalkyl, carbocyclylalkyl, alkynyl, alkenyl, oralkyl, or two R taken together may form a 4- to 8-membered ring, e.g.,with N;

Cy and Cy′ independently represent substituted or unsubstituted aryl,heterocyclyl, heteroaryl, or cycloalkyl, including polycyclic groups;

i represents, independently for each occurrence, an integer from 0 to 5,preferably from 0 to 2; and

k represents an integer from 0 to 3, preferably from 0 to 2.

In certain embodiments, NR₂ represents a primary amine or a secondary ortertiary amine substituted with one or two lower alkyl groups, arylgroups, or aralkyl groups, respectively, preferably a primary orsecondary amine.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Ar and Z independently represent substituted orunsubstituted aryl or heteroaryl rings, e.g., unsubstituted orsubstituted with one or more groups optionally including heteroatomssuch as O, N, and S. In certain embodiments, at least one of Ar and Zrepresents a phenyl ring. In certain embodiments, at least one of Ar andZ represents a heteroaryl ring, e.g., a pyridyl, thiazolyl, thienyl,pyrimidyl, furanyl, etc. In certain embodiments, the occurrences of Yattached to Ar are disposed in a meta and/or 1,3-relationship.

In certain embodiments, Y is absent from all positions. In certainembodiments, the only present occurrence of Y is attached to M_(k). Inembodiments wherein Y is present in a position, i or k preferablyrepresents 2 in an adjacent M_(i/k) if i/k=0 would result in twooccurrences of Y being directly attached to each other, or an occurrenceof Y being directly attached to N. In certain embodiments, where twooccurrences of Y are attached to M, at least one such occurrence of Y isabsent. In certain embodiments, no more than two occurrences of Y arepresent.

In certain embodiments, Cy′ is a substituted or unsubstituted aryl orheteroaryl. In certain embodiments, Cy′ is directly attached to X. Incertain embodiments, Cy′ is a substituted or unsubstituted bicyclic orheteroaryl ring, preferably both bicyclic and heteroaryl, such asbenzothiophene, benzofuran, benzopyrrole, benzopyridine, etc. In certainembodiments, Cy′ is a monocyclic aryl or heteroaryl ring substituted atleast with a substituted or unsubstituted aryl or heteroaryl ring, i.e.,forming a biaryl system. In certain embodiments, Cy′ includes twosubstituted or unsubstituted aryl or heteroaryl rings, e.g., the same ordifferent, directly connected by one or more bonds, e.g., to form abiaryl or bicyclic ring system. In certain embodiments, Cy′ represents abenzo(b)thien-2-yl, preferably a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl, e.g.,wherein the benzo ring is substituted with from 1-4 substituentsselected from halogen, nitro, cyano, methyl (e.g., including halomethyl,such as CHCl₂ and CF₃), and ethyl (e.g., including haloethyl, such asCH₂CCl₃, C₂F₅, etc.), preferably from halogen and methyl (e.g.,including halomethyl, such as CHCl₂ and CF₃). In certain suchembodiments Cy′ represents a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl wherein thebenzo ring is substituted with fluoro at the 4-position (peri to the3-substituent on the thienyl ring) and, optionally, at the 7-position(‘peri’ to the S of the thienyl ring).

In certain embodiments, Cy represents a substituted or unsubstitutednon-aromatic carbocyclic or heterocyclic ring, i.e., including at leastone sp³ hybridized atom, and preferably a plurality of sp³ hybridizedatoms. In certain embodiments, Cy is a 5- to 7-membered ring. In certainembodiments, Cy is directly attached to N and/or to NR₂. In embodimentswherein Cy is a six-membered ring directly attached to N and bears anamino substituent at the 4 position of the ring relative to N, the N andamine substituents may be disposed trans on the ring.

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, substituents on Ar or Z, where Z is an aryl orheteroaryl ring, are selected from halogen, lower alkyl, lower alkenyl,aryl, heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,acylamino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,—(CH₂)_(p)alkyl, —(CH₂)_(p)alkenyl, —(CH₂)_(p)alkynyl, —(CH₂)_(p)aryl,—(CH₂)_(p)aralkyl, —(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl,—(CH₂)_(p)O-lower alkenyl, —O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-loweralkyl, —(CH₂)_(p)S-lower alkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂,—(CH₂)_(p)NR-lower alkyl, —(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, andprotected forms of the above, wherein n and p, individually for eachoccurrence, represent integers from 0 to 10, preferably from 0 to 5.

In certain embodiments, Z is directly attached to Ar, or attached to Arthrough a chain of one or two atoms. In certain embodiments, Z-Y-M,taken together, is absent.

In certain embodiments, compounds useful in the present invention may berepresented by general formula (XII):

Formula XII

wherein, as valence and stability permit,

Ar represents a substituted or unsubstituted aryl or heteroaryl ring;

Z is absent or represents a substituted or unsubstituted aryl,carbocyclyl, heterocyclyl, or heteroaryl ring, or a lower alkyl, nitro,cyano, or halogen substituent;

Y, independently for each occurrence, is absent or represents —N(R)—,—O—, —S—, or —Se—, provided that if Z is not a ring, then Y attached toZ is absent;

X is selected from —C(═O)—, —C(═S)—, —S(O₂)—, —S(O)—, —C(═NCN)—,—P(═O)(OR)—, and a methylene group optionally substituted with 1-2groups such as lower alkyl, alkenyl, or alkynyl groups;

M represents, independently for each occurrence, a substituted orunsubstituted methylene group, such as —CH₂—, —CHF—, —CHOH—, —CH(Me)—,—C(═O)—, etc., or two M taken together represent substituted orunsubstituted ethene or ethyne;

R, independently for each occurrence, represents H or substituted orunsubstituted aryl, heterocyclyl, carbocyclyl, heteroaryl, aralkyl,heteroaralkyl, heterocyclylalkyl, carbocyclylalkyl, alkynyl, alkenyl, oralkyl;

Cy and Cy′ independently represent substituted or unsubstituted aryl,heterocyclyl, heteroaryl, or cycloalkyl, including polycyclic groups;and

k represents an integer from 0 to 1.

In certain embodiments, NR₂ represents a primary amine or a secondary ortertiary amine substituted with one or two lower alkyl groups,respectively, preferably a primary or secondary amine, most preferably asecondary amine.

In certain embodiments, M represents, independently for each occurrence,a substituted or unsubstituted methylene group, such as —CH₂—, —CHF—,—CHOH—, —CH(Me)—, —C(═O)—, etc.

In certain embodiments, Y is absent from all positions. In certainembodiments, where Y is adjacent to M_(k), either Y is absent or k=0. Incertain embodiments, for at least one occurrence of M_(k) attached toCy, k=0, optionally for both occurrences. In certain embodiments, forM_(k) attached to Ar and N, k=1.

In certain embodiments, Ar and Z independently represent substituted orunsubstituted aryl or heteroaryl rings, e.g., unsubstituted orsubstituted with one or more groups optionally including heteroatomssuch as O, N, and S. In certain embodiments, at least one of Ar and Zrepresents a phenyl ring. In certain embodiments, at least one of Ar andZ represents a heteroaryl ring, e.g., a pyridyl, thiazolyl, thienyl,pyrimidyl, furanyl, etc. In certain embodiments, the occurrences ofM_(k) attached to Ar are disposed in a meta and/or 1,3-relationship.

In certain embodiments, Cy′ is a substituted or unsubstituted aryl orheteroaryl. In certain embodiments, Cy′ is directly attached to X. Incertain embodiments, Cy′ is a substituted or unsubstituted bicyclic orheteroaryl ring, preferably both bicyclic and heteroaryl, such asbenzothiophene, benzofuran, benzopyrrole, benzopyridine, etc. In certainembodiments, Cy′ is a monocyclic aryl or heteroaryl ring substituted atleast with a substituted or unsubstituted aryl or heteroaryl ring, i.e.,forming a biaryl system. In certain embodiments, Cy′ includes twosubstituted or unsubstituted aryl or heteroaryl rings, e.g., the same ordifferent, directly connected by one or more bonds, e.g., to form abiaryl or bicyclic ring system. In certain embodiments, Cy′ represents abenzo(b)thien-2-yl, preferably a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl, e.g.,wherein the benzo ring is substituted with from 1-4 substituentsselected from halogen, nitro, cyano, methyl (e.g., including halomethyl,such as CHCl₂ and CF₃), and ethyl (e.g., including haloethyl, such asCH₂CCl₃, C₂F₅, etc.), preferably from halogen and methyl (e.g.,including halomethyl, such as CHCl₂ and CF₃). In certain suchembodiments Cy′ represents a 3-chloro-benzo(b)thien-2-yl,3-fluoro-benzo(b)thien-2-yl, or 3-methyl-benzo(b)thien-2-yl wherein thebenzo ring is substituted with fluoro at the 4-position (peri to the3-substituent on the thienyl ring) and, optionally, at the 7-position(‘peri’ to the S of the thienyl ring).

In certain embodiments, Cy represents a substituted or unsubstitutednon-aromatic carbocyclic or heterocyclic ring, i.e., including at leastone sp³ hybridized atom, and preferably a plurality of sp³ hybridizedatoms. In certain embodiments, Cy is a 5- to 7-membered ring. In certainembodiments, Cy is directly attached to N and/or to NR₂. In embodimentswherein Cy is a six-membered ring directly attached to N and bears anamino substituent at the 4 position of the ring relative to N, the N andamino substituents may be disposed trans on the ring.

In certain embodiments, X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.

In certain embodiments, substituents on Ar or Z, where Z is an aryl orheteroaryl ring, are selected from halogen, lower alkyl, lower alkenyl,aryl, heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,acylamino, cyano, nitro, hydroxyl, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, —(CH₂)_(p)alkyl, —(CH₂)_(p)alkenyl,—(CH₂)_(p)alkynyl, —(CH₂)_(p)OH, —(CH₂)_(p)O-lower alkyl,—(CH₂)_(p)O-lower alkenyl, —O(CH₂)_(n)R, —(CH₂)_(p)SH, —(CH₂)_(p)S-loweralkyl, —(CH₂)_(p)S-lower alkenyl, —S(CH₂)_(n)R, —(CH₂)_(p)N(R)₂,—(CH₂)_(p)NR-lower alkyl, —(CH₂)_(p)NR-lower alkenyl, —NR(CH₂)_(n)R, andprotected forms of the above, wherein n and p, individually for eachoccurrence, represent integers from 0 to 10, preferably from 0 to 5.

In certain embodiments, Z is directly attached to Ar, or attached to Arthrough a chain of one or two atoms. In certain embodiments, Z-Y-M,taken together, is absent.

In certain embodiments, the subject agonists can be chosen on the basisof their selectively for the hedgehog pathway. This selectivity can befor the hedgehog pathway versus other pathways, or for selectivitybetween particular hedgehog pathways, e.g., ptc-1, ptc-2, etc.

In certain preferred embodiments, the subject agonists modulate ptc-smomediated signal transduction with an ED₅₀ of 1 mM or less, morepreferably of 1 μM or less, and even more preferably of 1 nM or less.For hedgehog-dependent agonists, the subject agonists increase theactivity of hedgehog 10-fold, 100-fold, or even 1000-fold.

In particular embodiments, the small molecule is chosen for use becauseit is more selective for one patched isoform over the next, e.g.,10-fold, and more preferably at least 100- or even 1000-fold moreselective for one patched pathway (ptc-1, ptc-2) over another.

In certain embodiments, a compound which is an agonist of the hedgehogpathway is chosen to selectively agonize hedgehog activity over proteinkinases other than PKA, such as PKC, e.g., the compound modulates theactivity of the PKA/hedgehog pathway at least an order of magnitude morestrongly than it modulates the activity of another protein kinase,preferably at least two orders of magnitude more strongly, even morepreferably at least three orders of magnitude more strongly. Thus, forexample, a preferred activator of the hedgehog pathway may activatehedgehog activity with a K_(i) at least an order of magnitude lower thanits K_(i) for activation of PKC, preferably at least two orders ofmagnitude lower, even more preferably at least three orders of magnitudelower. In certain embodiments, the K_(i) for PKA/hedgehog activation isless than 10 nM, preferably less than 1 nM, even more preferably lessthan 0.1 nM.

Synthesis of Subject Compounds

Compounds of the present invention can be readily prepared by standardtechniques of organic synthesis, e.g., according to examples set forthin the Exemplification below. For example, a subject compound may beprepared by reacting a compound or pair of compounds designated A with acompound or pair of compounds designated B and a compound designated C,as set forth below:

Similarly, a compound designated C above may be reacted with a compoundor pair of compounds designated D and a compound or pair of compoundsdesignated E:

Alternatively, a compound or pair of compounds designated A above and acompound or pair of compounds designated E above may be reacted with acompound designated F:

Combinations of compounds as indicated above are preferably reacted witheach other in series, e.g., two compounds are reacted together, theproduct is reacted with a third compound, etc., and the compounds cangenerally be coupled in series in any order, as will be understood byone of skill in the art. In certain embodiments, functional groups onone or more compounds may require protection during one or morereactions, as is well understood in the art, and any suitable protectinggroups can be employed for this purpose. One of skill in the art canreadily select suitable protecting groups for a particular functionalgroup and a particular reaction. Elaboration steps may be performed atany time to modify functional groups or moieties on the product of areaction, for example, to convert N(R)₂═NH₂ to N(R)₂═NHR, e.g., bynucleophilic substitution, reductive alkylation, or any other suitablemethod.

In the compounds designated A-F above, the elements M, X, Y, Z, Cy, Cy′,Ar, i, k, R, etc. are defined as above (as may be broadened by thedescription below), and R′ independently for each occurrence representsH, a protecting group, or a labile reactive group, such as atrialkylsilyl (e.g., trimethylsilyl) group, and R″ independently foreach occurrence represents 1) a leaving group, such as a halogen (e.g.,F, Cl, Br, or I), alkylthio, cyano, alkoxy, or any other group capableof being replaced by an amine nucleophile when attached to X, 2) anactivatable group, such as OH, that can be activated by an activatingagent, such as a carbodiimide (e.g., diisopropylcarbodiimide,dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,etc.), phosphorous-based reagents (such as BOP-Cl, PyBROP, etc.), oxalylchloride, phosgene, triphosgene, or any similar reagent, to result in areactive intermediate having an increased susceptibility, relative tothe compound wherein R″═OH, towards coupling with an amine, or 3) X andR″ taken together represent an electrophilic group capable of reactingwith an amine, such as an isocyanate, isothiocyanate, or other similarreactive moiety.

The various subunits designated A-F can be combined using any of aplethora of reactions well known to those of skill in the art, dependingon the particular moieties to be coupled. For example, an amine, such asone of the NH₂ groups indicated on the subunits A-F, can be coupled withan alkyl group by reductive alkylation (e.g., the terminal occurrence ofM is an aldehyde), by nucleophilic displacement of a leaving group (suchas a halogen, sulfonate, or other suitable substituent), by nucleophilicopening of an epoxide, or by any other suitable reaction known to thoseof skill in the art. Similarly, amines can be coupled with activatedcarboxylic acid derivatives or thiocarboxylic acid derivatives, e.g.,prepared in situ from a carboxylic acid or thiocarboxylic acid and anactivating agent or prepared as isolated compounds such as isocyanates,carboxylic acid chlorides, etc., to provide amides, ureas, thioureas,thioamides, etc., with chloroformate esters, sulfonyl chlorides, orother such compounds to provide urethanes, sulfonamides, etc., or withother electrophilic reagents that form a covalent bond with an amine.

Aryl and/or heteroaryl rings can be readily coupled directly usingStille, Suzuki, or other related reactions, such as palladium-mediatedcross-coupling reactions. Aryl and/or heteroaryl rings can be readilycoupled through a heteroatom, e.g., using reactions such as the Ullmanreaction, any of various palladium-mediated reactions developed by S.Buchwald and others, by nucleophilic aromatic substitution, or othersuch reactions. Similarly, amines, alcohols, thiols, and other suchheteroatom-bearing compounds can be coupled to aryl and/or heteroarylrings using palladium-mediated reactions developed by S. Buchwald andothers, nucleophilic aromatic substitution, etc. Aryl and/or heteroarylrings linked by substituted or unsubstituted hydrocarbon chains can beprepared by Stille, Suzuki, Heck, Friedel-Crafts, and other reactions aswill be apparent to those of skill in the art.

A survey of a number of common synthetic reactions potentially usefulfor preparing compounds of the present invention are described ingreater detail below and in FIGS. 1-31. The variable groups included inthe subunits designated A-F above can be varied to correspond with anyof the Formulae I-VIII and X-XII without departing from the generalsynthesis approaches outlined above.

Similarly, compounds of the present invention can be prepared bycoupling a suitable moiety to a partially assembled structure. Forexample, a compound of Formula XI can be prepared by any of the stepsI-VI shown in the scheme below.

Similarly, a compound of Formula X may be prepared by any of the stepsin the scheme below.

By analogy, a compound of Formula XII can be prepared by any of thesteps set forth in the scheme below.

In the schemes above, M, Cy, Ar, X, Cy′, Y, Z, R, i, k, R′, and R″correspond to their use above, and can be more narrowly defined as setforth in the description of Formulae X-XII, or to correspond to elementsof Formulae I-VIII, optionally as modified by the descriptionaccompanying Formulae I-VIII.

Reactions suitable for performing Step I include palladium-mediatedreactions developed by S. Buchwald and others, nucleophilic aromaticsubstitution, oxidative coupling, etc.

Reactions suitable for performing Step II include nucleophilicdisplacement of a leaving group on M, reductive alkylation, reaction ofthe amine with an electrophilic carboxylic/thiocarboxylic acidderivative (acid chloride, isocyanate, isothiocyanate, or a carboxylicacid activated by BOP-Cl, PyBrOP, carbodiimide, or another activatingreagent (such as are commonly used in the art of peptide coupling)), orother similar reactions, including those set forth in detail in certainof FIGS. 1-31 and the accompanying description below, or, where M and Yare absent, a palladium-mediated coupling as developed by Buchwald andothers.

Reactions suitable for performing Steps III or IV include reaction ofY—R′ with an electrophilic carbonyl or sulfonyl derivative (X—R″=acidchloride, isocyanate, isothiocyanate, chloroformate, sulfonyl chloride,or an acid activated by BOP-Cl, PyBrOP, carbodiimide, or anotheractivating reagent (such as are commonly used in the art of peptidecoupling)), or other similar reactions, such as those set forth indetail in certain of FIGS. 1-31 and the accompanying description below.

Reactions suitable for performing Step V include nucleophilicdisplacement of a leaving group, reductive alkylation, reaction of theamine with an electrophilic carboxylic/thiocarboxylic acid derivative(acid chloride, isocyanate, isothiocyanate, or a carboxylic acidactivated by BOP-Cl, PyBrOP, carbodiimide, or another activating reagent(such as are commonly used in the art of peptide coupling)), or othersimilar reactions, including those set forth in detail in certain ofFIGS. 1-31 and the accompanying description below.

Reactions suitable for performing Step VI include nucleophilicdisplacement of a leaving group, reductive alkylation, reaction of theamine with an electrophilic carboxylic/thiocarboxylic acid derivative(acid chloride, isothiocyanate, isocyanate, or a carboxylic acidactivated by BOP-Cl, PyBrOP, carbodiimide, or another activating reagent(such as are commonly used in the art of peptide coupling)), or othersimilar reactions, including those set forth in detail in certain ofFIGS. 1-31 and the accompanying description below.

Reactions suitable for performing Step VII where Y is coupled with apresent occurrence of M include nucleophilic displacement of a leavinggroup, reductive alkylation, reaction of the amine with an electrophiliccarboxylic/thiocarboxylic acid derivative (acid chloride, isocyanate,isothiocyanate, or a carboxylic acid activated by BOP-Cl, PyBrOP,carbodiimide, or another activating reagent (such as are commonly usedin the art of peptide coupling)), or other similar reactions, includingthose set forth in detail in certain of FIGS. 1-31 and the accompanyingdescription below. In embodiments where occurrences of M is absent,suitable coupling reactions include palladium-mediated reactionsdeveloped by S. Buchwald and others, nucleophilic aromatic substitution,oxidative coupling, etc. In embodiments where M and Y are absent and Zrepresents an aryl or heteroaryl ring, suitable reactions includeStille, Suzuki, and other reactions suitable for forming biaryl systems.

Methods of the invention further include reacting a compound of any ofFormulae I-VII wherein at least one R of NR₂ represents H underconditions which convert that compound to a compound of the same formulawherein the corresponding occurrence of R represents a lower alkylgroup. For example, reductive alkylations with an aldehyde and areducing agent, nucleophilic alkylations with an alkyl halide such asMeI, or other similar reactions may be employed. In certain embodiments,such reactions may proceed through a silylated (e.g., R═SiMe₃)intermediate.

One of skill in the art will readily appreciate that compounds of thepresent invention are amenable to synthesis according to a wide array ofprotocols well known in the art in addition to those described herein,all of which are intended to fall within the scope of the presentinvention.

IV. Exemplary Applications of Method and Compositions

Another aspect of the present invention relates to a method ofmodulating a differentiated state, survival, and/or proliferation of acell, by contacting the cell with a hedgehog agonist according to thesubject method and as the circumstances may warrant.

For instance, it is contemplated by the invention that, in light of thefindings of an apparently broad involvement of hedgehog, ptc, andsmoothened in the formation of ordered spatial arrangements ofdifferentiated tissues in vertebrates, the subject method could be usedas part of a process for generating and/or maintaining an array ofdifferent vertebrate tissue both in vitro and in vivo. The hedgehogagonist, whether inductive or anti-inductive with respect proliferationor differentiation of a given tissue, can be, as appropriate, any of thepreparations described above.

For example, the present method is applicable to cell culturetechniques. In vitro neuronal culture systems have proved to befundamental and indispensable tools for the study of neural development,as well as the identification of neurotrophic factors such as nervegrowth factor (NGF), ciliary trophic factors (CNTF), and brain derivedneurotrophic factor (BDNF). One use of the present method may be incultures of neuronal stem cells, such as in the use of such cultures forthe generation of new neurons and glia. In such embodiments of thesubject method, the cultured cells can be contacted with a hedgehogagonist of the present invention in order to alter the rate ofproliferation of neuronal stem cells in the culture and/or alter therate of differentiation, or to maintain the integrity of a culture ofcertain terminally differentiated neuronal cells. In an exemplaryembodiment, the subject method can be used to culture, for example,sensory neurons or, alternatively, motor neurons. Such neuronal culturescan be used as convenient assay systems as well as sources ofimplantable cells for therapeutic treatments.

According to the present invention, large numbers of non-tumorigenicneural progenitor cells can be perpetuated in vitro and their rate ofproliferation and/or differentiation can be affected by contact withhedgehog agonists of the present invention. Generally, a method isprovided comprising the steps of isolating neural progenitor cells froman animal, perpetuating these cells in vitro or in vivo, preferably inthe presence of growth factors, and regulating the differentiation ofthese cells into particular neural phenotypes, e.g., neurons and glia,by contacting the cells with a hedgehog agonist.

Progenitor cells are thought to be under a tonic inhibitory influencewhich maintains the progenitors in a suppressed state until theirdifferentiation is required. However, recent techniques have beenprovided which permit these cells to proliferate, and unlike neuronswhich are terminally differentiated and therefore non-dividing, they canbe produced in unlimited number and are highly suitable fortransplantation into heterologous and autologous hosts withneurodegenerative diseases.

By “progenitor” it is meant an oligopotent or multipotent stem cellwhich is able to divide without limit and, under specific conditions,can produce daughter cells which terminally differentiate such as intoneurons and glia. These cells can be used for transplantation into aheterologous or autologous host. By heterologous is meant a host otherthan the animal from which the progenitor cells were originally derived.By autologous is meant the identical host from which the cells wereoriginally derived.

Cells can be obtained from embryonic, post-natal, juvenile or adultneural tissue from any animal. By any animal is meant any multicellularanimal which contains nervous tissue. More particularly, is meant anyfish, reptile, bird, amphibian or mammal and the like. The mostpreferable donors are mammals, especially mice and humans.

In the case of a heterologous donor animal, the animal may beeuthanized, and the brain and specific area of interest removed using asterile procedure. Brain areas of particular interest include any areafrom which progenitor cells can be obtained which will serve to restorefunction to a degenerated area of the host's brain. These regionsinclude areas of the central nervous system (CNS) including the cerebralcortex, cerebellum, midbrain, brainstem, spinal cord and ventriculartissue, and areas of the peripheral nervous system (PNS) including thecarotid body and the adrenal medulla. More particularly, these areasinclude regions in the basal ganglia, preferably the striatum whichconsists of the caudate and putamen, or various cell groups such as theglobus pallidus, the subthalamic nucleus, the nucleus basalis which isfound to be degenerated in Alzheimer's disease patients, or thesubstantia nigra pars compacta which is found to be degenerated inParkinson's disease patients.

Human heterologous neural progenitor cells may be derived from fetaltissue obtained from elective abortion, or from a post-natal, juvenileor adult organ donor. Autologous neural tissue can be obtained bybiopsy, or from patients undergoing neurosurgery in which neural tissueis removed, in particular during epilepsy surgery, and more particularlyduring temporal lobectomies and hippocampalectomies.

Cells can be obtained from donor tissue by dissociation of individualcells from the connecting extracellular matrix of the tissue.Dissociation can be obtained using any known procedure, includingtreatment with enzymes such as trypsin, collagenase and the like, or byusing physical methods of dissociation such as with a blunt instrumentor by mincing with a scalpel to a allow outgrowth of specific cell typesfrom a tissue. Dissociation of fetal cells can be carried out in tissueculture medium, while a preferable medium for dissociation of juvenileand adult cells is artificial cerebral spinal fluid (aCSF). Regular aCSFcontains 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃,and 10 mM D-glucose. Low Ca²⁺ aCSF contains the same ingredients exceptfor MgCl₂ at a concentration of 3.2 mM and CaCl₂ at a concentration of0.1 mM.

Dissociated cells can be placed into any known culture medium capable ofsupporting cell growth, including MEM, DMEM, RPMI, F-12, and the like,containing supplements which are required for cellular metabolism suchas glutamine and other amino acids, vitamins, minerals and usefulproteins such as transferrin and the like. Medium may also containantibiotics to prevent contamination with yeast, bacteria and fungi suchas penicillin, streptomycin, gentamicin and the like. In some cases, themedium may contain serum derived from bovine, equine, chicken and thelike. A particularly preferable medium for cells is a mixture of DMEMand F-12.

Conditions for culturing should be close to physiological conditions.The pH of the culture media should be close to physiological pH,preferably between pH 6-8, more preferably close to pH 7, even moreparticularly about pH 7.4. Cells should be cultured at a temperatureclose to physiological temperature, preferably between 30° C.-40° C.,more preferably between 32° C.-38° C., and most preferably between 35°C.-37° C.

Cells can be grown in suspension or on a fixed substrate, butproliferation of the progenitors is preferably done in suspension togenerate large numbers of cells by formation of “neurospheres” (see, forexample, Reynolds et al. (1992) Science 255:1070-1709; and PCTPublications WO93/01275, WO94/09119, WO94/10292, and WO94/16718). In thecase of propagating (or splitting) suspension cells, flasks are shakenwell and the neurospheres allowed to settle on the bottom corner of theflask. The spheres are then transferred to a 50 ml centrifuge tube andcentrifuged at low speed. The medium is aspirated, the cells resuspendedin a small amount of medium with growth factor, and the cellsmechanically dissociated and resuspended in separate aliquots of media.

Cell suspensions in culture medium are supplemented with any growthfactor which allows for the proliferation of progenitor cells and seededin any receptacle capable of sustaining cells, though as set out above,preferably in culture flasks or roller bottles. Cells typicallyproliferate within 3-4 days in a 37° C. incubator, and proliferation canbe reinitiated at any time after that by dissociation of the cells andresuspension in fresh medium containing growth factors.

In the absence of substrate, cells lift off the floor of the flask andcontinue to proliferate in suspension forming a hollow sphere ofundifferentiated cells. After approximately 3-10 days in vitro, theproliferating clusters (neurospheres) are fed every 2-7 days, and moreparticularly every 2-4 days by gentle centrifugation and resuspension inmedium containing growth factor.

After 6-7 days in vitro, individual cells in the neurospheres can beseparated by physical dissociation of the neurospheres with a bluntinstrument, more particularly by triturating the neurospheres with apipette. Single cells from the dissociated neurospheres are suspended inculture medium containing growth factors, and differentiation of thecells can be control in culture by plating (or resuspending) the cellsin the presence of a hedgehog agonist.

To further illustrate other uses of the subject hedgehog agonists, it isnoted that intracerebral grafting has emerged as an additional approachto central nervous system therapies. For example, one approach torepairing damaged brain tissues involves the transplantation of cellsfrom fetal or neonatal animals into the adult brain (Dunnett et al.(1987) J Exp Biol 123:265-289; and Freund et al. (1985) J Neurosci5:603-616). Fetal neurons from a variety of brain regions can besuccessfully incorporated into the adult brain, and such grafts canalleviate behavioral defects. For example, movement disorder induced bylesions of dopaminergic projections to the basal ganglia can beprevented by grafts of embryonic dopaminergic neurons. Complex cognitivefunctions that are impaired after lesions of the neocortex can also bepartially restored by grafts of embryonic cortical cells. The subjectmethod can be used to regulate the growth state in the culture, or wherefetal tissue is used, especially neuronal stem cells, can be used toregulate the rate of differentiation of the stem cells.

Stem cells useful in the present invention are generally known. Forexample, several neural crest cells have been identified, some of whichare multipotent and likely represent uncommitted neural crest cells, andothers of which can generate only one type of cell, such as sensoryneurons, and likely represent committed progenitor cells. The role ofhedgehog agonists employed in the present method to culture such stemcells can be to regulate differentiation of the uncommitted progenitor,or to regulate further restriction of the developmental fate of acommitted progenitor cell towards becoming a terminally differentiatedneuronal cell. For example, the present method can be used in vitro toregulate the differentiation of neural crest cells into glial cells,schwann cells, chromaffin cells, cholinergic sympathetic orparasympathetic neurons, as well as peptidergic and serotonergicneurons. The hedgehog agonists can be used alone, or can be used incombination with other neurotrophic factors which act to moreparticularly enhance a particular differentiation fate of the neuronalprogenitor cell.

In addition to the implantation of cells cultured in the presence of thesubject hedgehog agonists, yet another aspect of the present inventionconcerns the therapeutic application of a hedgehog agonist to regulatethe growth state of neurons and other neuronal cells in both the centralnervous system and the peripheral nervous system. The ability of ptc,hedgehog, and smoothened to regulate neuronal differentiation duringdevelopment of the nervous system and also presumably in the adult stateindicates that, in certain instances, the subject hedgehog agonists canbe expected to facilitate control of adult neurons with regard tomaintenance, functional performance, and aging of normal cells; repairand regeneration processes in chemically or mechanically lesioned cells;and treatment of degeneration in certain pathological conditions. Inlight of this understanding, the present invention specificallycontemplates applications of the subject method to the treatmentprotocol of (prevention and/or reduction of the severity of)neurological conditions deriving from: (i) acute, subacute, or chronicinjury to the nervous system, including traumatic injury, chemicalinjury, vascular injury and deficits (such as the ischemia resultingfrom stroke), together with infectious/inflammatory and tumor-inducedinjury; (ii) aging of the nervous system including Alzheimer's disease;(iii) chronic neurodegenerative diseases of the nervous system,including Parkinson's disease, Huntington's chorea, amyotrophic lateralsclerosis and the like, as well as spinocerebellar degenerations; and(iv) chronic immunological diseases of the nervous system or affectingthe nervous system, including multiple sclerosis. For example, in thespecific case of Parkinson's disease, intervention by increasing theactivity of hedgehog by a subject agonist can improve the in vivosurvival of fetal and adult dopaminergic neurons, and thus can provide amore effective treatment of this disease. Thus, in one embodiment, thesubject method comprises administering to an animal afflicted withParkinson's disease, or at risk of developing Parkinson's disease, anamount of a hedgehog agonist effective for increasing the rate ofsurvival of dopaminergic neurons in the animal.

The present method is applicable to cell culture techniques. In vitroneuronal culture systems have proved to be fundamental and indispensabletools for the study of neural development, as well as the identificationof neurotrophic factors such as nerve growth factor (NGF), ciliarytrophic factors (CNTF), and brain derived neurotrophic factor (BDNF).Once a neuronal cell has become terminally differentiated it typicallywill not change to another terminally differentiated cell-type. However,neuronal cells can nevertheless readily lose their differentiated state.This is commonly observed when they are grown in culture from adulttissue, and when they form a blastema during regeneration. The presentmethod provides a means for ensuring an adequately restrictiveenvironment in order to maintain dopaminergic and GABAergic cells indifferentiated states, and can be employed, for instance, in cellcultures designed to test the specific activities of other trophicfactors.

In such embodiments of the subject method, a culture of differentiatedcells including dopaminergic and/or GABAergic cells can be contactedwith a hedgehog agonist in order to maintain the integrity of a cultureof terminally differentiated neuronal cells by preventing loss ofdifferentiation. The subject method can be used in conjunction withagents which induce the differentiation of neuronal precursors, e.g.,progenitor or stem cells, into dopaminergic or GABAergic neurons.

Many neurological disorders are associated with degeneration of discretepopulations of neuronal elements and may be treatable with a therapeuticregimen which includes a hedgehog agonist. For example, Alzheimer'sdisease is associated with deficits in several neurotransmitter systems,both those that project to the neocortex and those that reside with thecortex. For instance, the nucleus basalis in patients with Alzheimer'sdisease have been observed to have a profound (75%) loss of neuronscompared to age-matched controls. Although Alzheimer's disease is by farthe most common form of dementia, several other disorders can producedementia. Several of these are degenerative diseases characterized bythe death of neurons in various parts of the central nervous system,especially the cerebral cortex. However, some forms of dementia areassociated with degeneration of the thalamus or the white matterunderlying the cerebral cortex. Here, the cognitive dysfunction resultsfrom the isolation of cortical areas by the degeneration of efferentsand afferents. Huntington's disease involves the degeneration ofintrastriatal and cortical cholinergic neurons and GABAergic neurons.Pick's disease is a severe neuronal degeneration in the neocortex of thefrontal and anterior temporal lobes, sometimes accompanied by death ofneurons in the striatum. Treatment of patients suffering from suchdegenerative conditions can include the application of hedgehog agonistsin order to control, for example, differentiation and apoptotic eventswhich give rise to loss of neurons (e.g., to enhance survival ofexisting neurons) as well as promote differentiation and repopulation byprogenitor cells in the area affected.

In addition to degenerative-induced dementias, a pharmaceuticalpreparation of one or more of the subject hedgehog agonists can beapplied opportunely in the treatment of neurodegenerative disorderswhich have manifestations of tremors and involuntary movements.Parkinson's disease, for example, primarily affects subcorticalstructures and is characterized by degeneration of the nigrostriatalpathway, raphe nuclei, locus cereleus, and the motor nucleus of vagus.Ballism is typically associated with damage to the subthalmic nucleus,often due to acute vascular accident. Also included are neurogenic andmyopathic diseases which ultimately affect the somatic division of theperipheral nervous system and are manifest as neuromuscular disorders.Examples include chronic atrophies such as amyotrophic lateralsclerosis, Guillain-Barre syndrome and chronic peripheral neuropathy, aswell as other diseases which can be manifest as progressive bulbarpalsies or spinal muscular atrophies. The present method is amenable tothe treatment of disorders of the cerebellum which result in hypotoniaor ataxia, such as those lesions in the cerebellum which producedisorders in the limbs ipsilateral to the lesion. For instance, apreparation of a hedgehog agonist can used to treat a restricted form ofcerebellar cortical degeneration involving the anterior lobes (vermisand leg areas) such as is common in alcoholic patients.

In an illustrative embodiment, the subject method is used to treatamyotrophic lateral sclerosis. ALS is a name given to a complex ofdisorders that comprise upper and lower motor neurons. Patients maypresent with progressive spinal muscular atrophy, progressive bulbarpalsy, primary lateral sclerosis, or a combination of these conditions.The major pathological abnormality is characterized by a selective andprogressive degeneration of the lower motor neurons in the spinal cordand the upper motor neurons in the cerebral cortex. The therapeuticapplication of a hedgehog agonist can be used alone, or in conjunctionwith other neurotrophic factors such as CNTF, BDNF or NGF to preventand/or reverse motor neuron degeneration in ALS patients.

Hedgehog agonists of the present invention can also be used in thetreatment of autonomic disorders of the peripheral nervous system, whichinclude disorders affecting the enervation of smooth muscle andendocrine tissue (such as glandular tissue). For instance, the subjectmethod can be used to treat tachycardia or atrial cardiac arrhythmiaswhich may arise from a degenerative condition of the nerves innervatingthe striated muscle of the heart.

Furthermore, a potential role for certain of the hedgehog agonistsderives from the role of hedgehog proteins in development andmaintenance of dendritic processes of axonal neurons. Potential rolesfor hedgehog agonists consequently include guidance for axonalprojections and the ability to promote differentiation and/ormaintenance of the innervating cells to their axonal processes.Accordingly, compositions comprising hedgehog agonists may be employedto support the survival and reprojection of several types of ganglionicneurons sympathetic and sensory neurons as well as motor neurons. Inparticular, such therapeutic compositions may be useful in treatmentsdesigned to rescue, for example, various neurons from lesion-induceddeath as well as guiding reprojection of these neurons after suchdamage. Such diseases include, but are not limited to, CNS traumainfarction, infection (such as viral infection with varicella-zoster),metabolic disease, nutritional deficiency, toxic agents (such ascisplatin treatment).

As appropriate, the subject method can also be used in generating nerveprostheses for the repair of central and peripheral nerve damage. Inparticular, where a crushed or severed axon is intubulated by use of aprosthetic device, hedgehog agonists can be added to the prostheticdevice to regulate the rate of growth and regeneration of the dendriticprocesses. Exemplary nerve guidance channels are described in U.S. Pat.Nos. 5,092,871 and 4,955,892.

In another embodiment, the subject method can be used in the treatmentof neoplastic or hyperplastic transformations such as may occur in thecentral nervous system. For instance, the hedgehog agonists can beutilized to cause such transformed cells to become either post-mitoticor apoptotic. The present method may, therefore, be used as part of atreatment for, e.g., malignant gliomas, meningiomas, medulloblastomas,neuroectodermal tumors, and ependymomas.

The subject method has wide applicability to the treatment orprophylaxis of disorders affecting the regulation of peripheral nerves,including peripheral ganglionic neurons, sympathetic, sensory neurons,and motor neurons. In general, the method can be characterized asincluding a step of administering to an animal an amount of a hedgehogagonist effective to alter the proliferative and/or differentiationstate of treated peripheral nerve cells. Such therapeutic compositionsmay be useful in treatments designed to rescue, for example, retinalganglia, inner ear and acoustical nerves, and motor neurons, fromlesion-induced death as well as guiding reprojection of these neuronsafter such damage. Such diseases and conditions include, but are notlimited to, chemical or mechanical trauma, infection (such as viralinfection with varicella-zoster), metabolic disease such as diabetes,nutritional deficiency, and toxic agents (such as cisplatin treatment).The goals of treatment in each case can be twofold: (1) to eliminate thecause of the disease and (2) to relieve its symptoms.

Peripheral neuropathy is a condition involving nerve-ending damage inthe hands and feet. Peripheral neuropathy generally refers to a disorderthat affects the peripheral nerves, most often manifested as one or acombination of motor, sensory, sensorimotor, or autonomic neuraldysfunction. The wide variety of morphologies exhibited by peripheralneuropathies can each be uniquely attributed to an equally wide varietyof causes. For instance, peripheral neuropathies can be geneticallyacquired, can result from a systemic disease, or can be induced by atoxic agent. Some toxic agents that cause neurotoxicities aretherapeutic drugs, antineoplastic agents, contaminants in foods ormedicinals, and environmental and industrial pollutants.

In particular, chemotherapeutic agents known to cause sensory and/ormotor neuropathies include vincristine, an antineoplastic drug used totreat hematological malignancies and sarcomas. The neurotoxicity isdose-related, and exhibits as reduced intestinal motility and peripheralneuropathy, especially in the distal muscles of the hands and feet,postural hypotension, and atony of the urinary bladder. Similar problemshave been documented with taxol and cisplatin (Mollman, J. E., 1990, NewEng Jour Med. 322:126-127), although cisplatin-related neurotoxicity canbe alleviated with nerve growth factor (NGF) (Apfel, S. C. et al, 1992,Annals of Neurology 31:76-80). Although the neurotoxicity is sometimesreversible after removal of the neurotoxic agent, recovery can be a veryslow process (Legha, S., 1986, Medical Toxicology 1:421-427; Olesen, etal., 1991, Drug Safety 6:302-314).

There are a number of inherited peripheral neuropathies, including:Refsum's disease, abetalipoproteinemia, Tangier disease, Krabbe'sdisease, metachromatic leukodystrophy, Fabry's disease, Dejerine-Sottassyndrome, and others. Of all the inherited neuropathies, the most commonby far is Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth (CMT) Disease (also known as peroneal muscularatrophy, or hereditary motor sensory neuropathy (HMSN)) is the mostcommon hereditary neurological disorder. It is characterized by weaknessand atrophy, primarily of the peroneal muscles, due to segmentaldemyelination of peripheral nerves and associated degeneration of axonsand anterior horn cells. Autosomal dominant inheritance is usual, andassociated degenerative CNS disorders, such as Friedreich's ataxia, arecommon.

In one aspect, the method of the present invention can be used in thetreatment and maintenance of hereditary neuropathies. This group ofneuropathies is now becoming increasingly recognized due to the dramaticadvances in molecular genetics. The symptoms of the various hereditaryneuropathies are wide-ranging. A common denominator is usually the earlyonset of mild numbness and tingling in the feet that slowly progressesto involve the legs and the hands and later the rest of the upperextremities. Most of the hereditary neuropathies do have a motorcomponent consisting of distal weakness in the lower and upperextremities. A majority of patients with hereditary neuropathies havehigh arches in their feet or other bony deformities. The symptoms arevery slowly progressive and the majority of the patients are stillwalking two decades after the onset of their symptoms.

The diagnosis of a hereditary neuropathy is usually suggested with theearly onset of neuropathic symptoms, especially when a positive familyhistory is also present. Prior to the recent genetic advances, thediagnosis was supported by typical findings of marked slowing of thenerve conduction studies on electromyography and a nerve biopsy. Typicalfindings on a nerve biopsy include the presence of so-calledonion-bulbs, indicating a recurring demyelinating and remyelinating ofthe nerve fibers. With the most recent genetic advances, two majorhereditary neuropathies known as Charcot-Marie-Tooth disease andhereditary neuropathy with liability to pressure palsies can bediagnosed with a simple blood test that identifies the differentmutations responsible for these two entities.

Hereditary neuropathies are caused by genetic abnormalities which aretransmitted from generation to generation. For several of these, thegenetic defect is known, and tests are available for diagnosis andprenatal counseling.

As set forth above, the subject method can be used as part of atherapeutic regimen in the treatment of Charcot-Marie Tooth Disease(CMT). This is a general term given to the hereditary sensorimotorneuropathies. CMT type 1 (CMT 1) is associated with demyelination orbreakdown of the myelin sheaths. Several different abnormalities havebeen identified. CMT Type 1A is most commonly caused by duplication of agene encoding a myelin protein called PMP-22, and CMT type 1B is causedby a mutation in a myelin protein called the Po glycoprotein. CMTX is ahereditary sensorimotor neuropathy which primarily affects men. It iscaused by a mutation in a gene encoding a protein called Connexin 32 onthe X-chromosome.

In another embodiment, the subject method can be used in the treatmentof familial amyloidotic neuropathy and other related hereditaryneuropathies. Amyloidotic neuropathy usually presents with pain, sensoryloss and autonomic dysfunction. It is caused by a mutation in a proteincalled Transthyretin, resulting in deposition of the protein as amyloidin the peripheral nerves.

The subject method can be used in the treatment of hereditary porphyria,which can have components of peripheral neuropathy. Still anotherhereditary neuropathy for which the subject methods can be used fortreatment is hereditary sensory neuropathy Type II (HSN II). The methodsand compositions of the present invention can also be used in thetreatment and maintenance of acquired neuropathies.

For example, hedgehog agonists can be used to prevent diabeticneuropathies. Diabetes is the most common known cause of neuropathy. Itproduces symptoms in approximately 10% of people with diabetes. In mostcases, the neuropathy is predominantly sensory, with pain and sensoryloss in the hands and feet. But some diabetics have mononeuritis ormononeuritis multiplex which causes weakness in one or more nerves, orlumbosacral plexopathy or amyotrophy which causes weakness in the legs.

The instant method can also be used in the treatment of immune-mediatedneuropathies. The main function of the immune system is to protect thebody against infectious organisms which enter from outside. In somecases, however the immune system turns against the body and causesautoimmune disease. The immune system consists of several types of whiteblood cells, including T-lymphocytes, which also regulate the immuneresponse; and B-lymphocytes or plasma cells, which secrete specializedproteins called “antibodies” Sometimes, for unknown reasons, the immunesystem mistakenly attacks parts of the body such as the peripheralnenes. This is “autoimmune” Peripheral Neuropathy. There are severaldifferent types, depending on the part of the peripheral nerve which isattacked and the type of the immune reaction. The following are briefdescriptions of the neuropathies which are mediated by the immunesystem.

For instance, a hedgehog agonist can be used to treat Guillain-Barresyndrome (GBS), an acute neuropathy that comes on suddenly or rapidly.Guillain-Barre syndrome can progress to paralysis and respiratoryfailure within days or weeks after onset. The neuropathy is caused whenthe immune system destroys the myelin sheaths of the motor and sensorynerves. It is often preceded by infection, vaccination or trauma, andthat is thought to be what triggers the autoimmune reaction. The diseaseis self-limiting, with spontaneous recovery within six to eight weeks.But the recovery is often incomplete.

Other neuropathies which begin acutely, and which can be treated by themethod of the present invention, include acute motor neuropathy, acutesensory neuropathy, and acute autonomic neuropathy, in which there is animmune attack against the motor, sensory or autonomic nerves,respectively. The Miller-Fisher syndrome is another variant in whichthere is paralysis of eye gaze, incoordination, and unsteady gait.

Still another acquired neuropathy which is may be treated by the subjectmethod is chronic inflammatory demyelinating polyneuropathy (CIDP). CIDPis thought to be a chronic and more indolent form of the Guillain-Barresyndrome. The disease progresses either with repeated attacks, calledrelapses, or in a stepwise or steady fashion. As in GBS, there appearsto be destruction of the myelin sheath by antibodies and T-lymphocytes.But since there is no specific test for CIDP, the diagnosis is based onthe clinical and laboratory characteristics.

Chronic polyneuropathies with antibodies to peripheral nerves is stillanother peripheral neuropathy for which the subject methods can beemployed to treat or prevent. In some types of chronic neuropathies,antibodies to specific components of nerve have been identified. Theseinclude demyelinating neuropathy associated with antibodies to themyelin associated glycoprotein (MAG), motor neuropathy associated withantibodies to the gangliosides GM1 or GD1a, and sensory neuropathyassociated with anti-sulfatide or GD1b ganglioside antibodies. Theantibodies in these cases bind to oligosaccharide or sugar likemolecules, which are linked to proteins (glycoproteins) or lipids(glycolipids or gangliosides) in the nerves. It is suspected that theseantibodies may be responsible for the neuropathies.

The subject method can also be used as part of a therapeutic plan fortreating neuropathies associated with vasculitis or inflammation of theblood vessels in peripheral nerves. Neuropathy can also be caused byvasculitis—an inflammation of the blood vessels in peripheral nerve. Itproduces small “strokes” along the course of the peripheral nerves, andmay be restricted to the nerves or it may be generalized, include a skinrash, or involve other organs. Several rheumatological diseases likerheumatoid arthritis, lupus, periarteritis nodosa, or Sjogren'ssyndrome, are associated with generalized vasculitis, which can alsoinvolve the peripheral nerves. Vasculitis can cause polyneuritis,mononeuritis, or mononeuritis multiplex, depending on the distributionand severity of the lesions.

In still another embodiment, the method of the present invention can beused for treatment of brachial or lumbosacral plexitis. The brachialplexus, which lies under the armpit, contains the nerves to the arm andhand. Brachial plexitis is the result of inflammation of that nervebundle, and produces weakness and pain in one or both arms. Umbosacralplexitis, which occurs in the pelvis, causes weakness and pain in thelegs.

Hedgehog agonists may also be suitable for use in the treatment ofneuropathies associated with monoclonal gammopathies. In monoclonalgammopathy, single clones of B-cells or plasma cells in the bone marrowor lymphoid organs expand to form benign or malignant tumors and secreteantibodies. “Monoclonal” is because there are single clones ofantibodies, and “gammopathy” stands for gammaglobulins, which is anothername for antibodies. In some cases, the antibodies react with nervecomponents; in others, fragments of the antibodies form amyloiddeposits.

Yet another aspect of the present invention relates to the use of thesubject method in the treatment of neuropathies associated with tumorsor neoplasms. Neuropathy can be due to direct infiltration of nerves bytumor cells or to indirect effect of the tumor. The latter is calledparaneoplastic neuropathy. Several types have been described. Forinstance, the subject methods can be used to manage sensory neuropathyassociated with lung cancer. This neuropathy is associated withantibodies to a protein called Hu, which is present in the sensoryneurons of the peripheral nerves. Likewise, the subject method can beused to treat neuropathies associated with multiple myeloma. Multiplemyeloma is a bony tumor which is caused by antibody-secreting plasmacells in the bone marrow. The tumor is made up of a single clone ofplasma cells, and the antibodies they produce are identical ormonoclonal. Some people with multiple myeloma develop sensorimotorpolyneuropathy with degeneration of axons in the peripheral nerves. Inother embodiments, the subject method can be used to treat neuropathiesassociated with Waldenstrom's macroglobulemia, chronic lymphocyticleukemia, or B-cell lymphoma. These are tumors caused byantibody-secreting B-lymphocytes in the spleen, bone marrow or lymphnodes. These antibodies are monoclonal and frequently react withperipheral nerve components such as MAG, GM1, or sulfatide. In stillother embodiments, the hedgehog agonists of the present invention can beused as part of therapeutic protocol for the treatment of patients withcancers where neuropathy is a consequence of local irradiation or becaused by medications such as vincristine and cisplatin.

The present invention also contemplates the use of hedgehog agonists forthe treatment of neuropathies associated with amyloidosis. Amyloid is asubstance deposited in the peripheral nerves and interferes with theiroperation: the disorder is amyloidosis. There are two main types:primary amyloidosis, in which the deposits contain fragments ofmonoclonal antibodies (see monoclonal gammopathy above); and hereditaryamyloidosis in which the deposits contain a mutated protein calledTransthyretin. Primary amyloidosis is usually associated with monoclonalgammopathies or myeloma.

Still another aspect of the present invention provides the subjectmethod as a means for treating neuropathies caused by infections.Peripheral neuropathies can be caused by infection of the peripheralnerves. Viruses that cause peripheral neuropathies include the AIDSvirus, HIV-I, which causes slowly progressive sensory neuropathy,Cytomegalovirus which causes a rapidly progressive paralytic neuropathy,Herpes zoster which cause shingles, and poliovirus which causes a motorneuropathy. Hepatitis B or C infections are sometimes associated withvasculitic neuropathy.

Bacterial infections that cause neuropathy include leprosy, which causesa patchy sensory neuropathy, and diphtheria which can cause a rapidlyprogressive paralytic neuropathy. Other infectious diseases that causeneuropathy include Lyme disease, which is caused by a spirochete, andtrypanosomiasis which is caused by a parasite. Both commonly presentwith a multifocal neuropathy

Neuropathies caused by nutritional imbalance are also candidatedisorders for treatment by the subject method. Deficiencies of vitaminsB12, B1 (thiamine), B6 (pyridoxine), or E, for example, can producepolyneuropathies with degeneration of peripheral nerve axons. This canbe due to poor diet, or inability to absorb the nutrients from thestomach or gut. Moreover, megadoses of vitamin B6 can also cause aperipheral neuropathy, and the subject method can be used as part of adetoxification program in such cases.

Yet another use of the subject method is in the treatment ofneuropathies arising in kidney diseases. Chronic renal failure can causea predominantly sensory peripheral neuropathy with degeneration ofperipheral nerve axons.

Another aspect of the present invention provides a method for treatinghypothyroid neuropathies. Hypothyroidism is sometimes associated with apainful sensory polyneuropathy with axonal degeneration. Mononeuropathyor mononeuropathy multiplex can also occur due to compression of theperipheral nerves by swollen tissues.

The subject method can also be used in the treatment of neuropathiescaused by alcohol and toxins. Certain toxins can cause peripheralneuropathy. Lead toxicity is associated with a motor neuropathy; arsenicor mercury cause a sensory neuropathy, and thallium can cause a sensoryand autonomic neuropathy. Several organic solvents and insecticides canalso cause polyneuropathy. Alcohol is directly toxic to nerves andalcohol abuse is a major cause of neuropathy. The subject method can beused, in certain embodiments, as part of a broader detoxificationprogram.

In still another embodiment, the methods and compositions of the presentinvention can be used for the treatment of neuropathies caused by drugs.Several drugs are known to cause neuropathy. They include, among others,vincristine and cisplatin in cancer, nitrofurantoin, which is used inpyelonephritis, amiodarone in cardiac arrhythmias, disulfiram inalcoholism, ddC and ddI in AIDS, and dapsone which is used to treatleprosy. As above, the subject method can be used, in certainembodiments, as part of a broader detoxification program.

The method of the present invention can also be used in the treatment ofneuropathies caused by trauma or compression. Localized neuropathies canresult from compression of nerves by external pressure or overlyingtendons and other tissues. The best known of these are the carpal tunnelsyndrome which results from compression at the wrist, and cervical orlumbar radiculopathies (sciatica) which result from compression of nerveroots as they exit the spine. Other common areas of nerve compressioninclude the elbows, armpits, and the back of the knees.

The subject method is also useful in variety of idiopathic neuropathies.The term “idiopathic” is used whenever the cause of the neuropathycannot be found. In these cases, the neuropathy is classified accordingto its manifestations, i.e., sensory, motor, or sensorimotor idiopathicpolyneuropathy.

The subject method has wide applicability to the treatment orprophylaxis of disorders afflicting muscle tissue. In general, themethod can be characterized as including a step of administering to ananimal an amount of a hedgehog agonist effective to alter theproliferative state of a treated muscle tissue. The mode ofadministration and dosage regimens will vary depending on the muscletissue(s) which is to be treated.

In one aspect, the invention is directed to a muscle-trophic factor, andits use in stimulating muscle growth or differentiation in mammals. Suchstimulation of muscle growth is useful for treating atrophy, or wasting,in particular, skeletal muscle atrophy and cardiac muscle atrophy. Inaddition, certain diseases wherein the muscle tissue is damaged, isabnormal or has atrophied, are treatable using the invention, such as,for example, normal aging, disuse atrophy, wasting or cachexia, andvarious secondary disorders associated with age and the loss of musclemass, such as hypertension, glucose intolerance and diabetes,dyslipidemia and atherosclerotic cardiovascular disease. The treatmentof muscular myopathies such as muscular dystrophies is also embodied inthe invention.

With denervation or disuse, skeletal muscles undergo rapid atrophy whichleads to a profound decrease in size, protein content and contractilestrength. This atrophy is an important component of many neuromusculardiseases in humans. In a clinical setting, compositions comprising thesubject hedgehog agonists can be used for inhibiting muscledegeneration, e.g., for decreasing the loss of muscle mass, such as partof a treatment for such muscle wasting disorders.

In preferred embodiments pharmaceutical compositions according to theinvention are administered to patients suffering from a disorder, i.e.,an abnormal physical condition, a disease or pathophysiologicalcondition associated with abnormal and/or aberrant regulation of muscletissue. The disorders for which the compositions of the invention areadministered are preferably those which directly or indirectly produce awasting (i.e., loss) of muscle mass, that is, a muscle wasting disorder.These include muscular dystrophies, cardiac cachexia, emphysema,leprosy, malnutrition, osteomalacia, child acute leukemia, AIDS cachexiaand cancer cachexia.

The muscular dystrophies are genetic diseases which are characterized byprogressive weakness and degeneration of muscle fibers without evidenceof neural degeneration. In Duchenne muscular dystrophy (DMD) patientsdisplay an average of a 67% reduction in muscle mass, and in myotonicdystrophy, fractional muscle protein synthesis has been shown to bedecreased by an average of 28%, without any corresponding decrease innon-muscle protein synthesis (possibly due to impaired end-organresponse to anabolic hormones or substrates). Accelerated proteindegradation has been demonstrated in the muscles of DMD patients. Thesubject method can be used as part of a therapeutic strategy forpreventing, and in some instance reversing, the muscle wastingconditions associated with such dystrophies.

Severe congestive heart failure (CHF) is characterized by a “cardiaccachexia,” i.e., a muscle protein wasting of both the cardiac andskeletal muscles, with an average 19% body weight decrease. The cardiaccachexia is caused by an increased rate of myofibrillar proteinbreakdown. The subject method can be used as part of a treatment forcardiac cachexia.

Emphysema is a chronic obstructive pulmonary disease, defined by anenlargement of the air spaces distal to the terminal non-respiratorybronchioles, accompanied by destructive changes of the alveolar walls.Clinical manifestations of reduced pulmonary functioning includecoughing, wheezing, recurrent respiratory infections, edema, andfunctional impairment and shortened lifespan. The efflux of tyrosine isincreased by 47% in emphysematous patients. Also, whole body leucineflux remains normal, whole-body leucine oxidation is increased, andwhole-body protein synthesis is decreased. The result is a decrease inmuscle protein synthesis, accompanied by a decrease in whole bodyprotein turnover and skeletal muscle mass. This decrease becomesincreasingly evident with disease progression and long-termdeterioration. The subject hedgehog agonists may be used to preventand/or reverse, the muscle wasting conditions associated with suchdiseases.

In diabetes mellitus, there is a generalized wasting of small muscle ofthe hands, which is due to chronic partial denervation (neuropathy).This is most evident and worsens with long-term disease progression andseverity. The subject method can be used as part of a therapeuticstrategy for treatment of diabetes mellitus.

Leprosy is associated with a muscular wasting which occurs between themetacarpals of the thumb and index finger. Severe malnutrition ischaracterized by, inter alia, severe muscle wasting. The subject methodcan be used to treat muscle-wasting effects of leprosy.

Osteomalacia is a nutritional disorder caused by a deficiency of vitaminD and calcium. It is referred to as “rickets” in children, and“osteomalacia” in adults. It is marked by a softening of the bones (dueto impaired mineralization, with excess accumulation of osteoid), pain,tenderness, muscle wasting and weakness, anorexia, and overall weightloss. It can result from malnutrition, repeated pregnancies andlactation (exhausting or depleting vitamin D and calcium stores), andvitamin D resistance. The subject method can be used as part of atherapeutic strategy for treatment of osteomalacia.

In childhood acute leukemia there is protein energy malnutrition whichresults in skeletal muscle wasting. Studies have shown that somechildren exhibit the muscle wasting even before diagnosis of theleukemia, with an average 27% decrease in muscle mass. There is also asimultaneous 33%-37% increase in adipose tissue, resulting in no netchange in relative body weight and limb circumference. Such patients maybe amenable to treatment with a hedgehog agonist according to the methodof the present invention.

Cancer cachexia is a complex syndrome which occurs with variableincidence in patients with solid tumors and hematological malignancies.Clinically, cancer cachexia is manifested as weight loss with massivedepletion of both adipose tissue and lean muscle mass, and is one causeof death which results from cancer. Cancer cachexia patients haveshorter survival times, and decreased response to chemotherapy. Inaddition to disorders which produce muscle wasting, other circumstancesand conditions appear to be linked in some fashion with a decrease inmuscle mass. Such afflictions include muscle wasting due to chronic backpain, advanced age, long-term hospitalization due to illness or injury,alcoholism and corticosteroid therapy. The subject method can be used aspart of a therapeutic strategy for preventing, and in some instancereversing, the muscle wasting conditions associated with such cancers.

Studies have shown that in severe cases of chronic lower back pain,there is paraspinal muscle wasting. Decreasing paraspinal muscle wastingalleviates pain and improves function. A course of treatment fordisorder can include administration of a therapeutic amount of ahedgehog agonist.

It is also believed that general weakness in old age is due to musclewasting. As the body ages, an increasing proportion of skeletal muscleis replaced by fibrous tissue. The result is a significant reduction inmuscle power, but only a marginal reduction in fat-free mass. Thesubject method can be used as part of a treatment and preventivestrategies for preventing/reversing muscle wasting in elderly patients.

Studies have also shown that in patients suffering injuries or chronicillnesses, and hospitalized for long periods of time, there islong-lasting unilateral muscle wasting, with an average 31% decrease inmuscle mass. Studies have also shown that this can be corrected withintensive physiotherapy. However, it may be more effective for manypatients to at least augment such therapies with treatment by thesubject method

In alcoholics there is wasting of the anterior tibial muscle. Thisproximal muscle damage is caused by neurogenic damage, namely, impairedglycolytic and phosphorylase enzyme activity. The damage becomesapparent and worsens the longer the duration of the alcohol abuse.Patients treated with corticosteroids experience loss of muscle mass.Such patients may also be amenable to treatment by the subject method.

The compounds of the invention can be used to alleviate the muscle massloss resulting from the foregoing conditions, as well as others.Additionally, the hedgehog agonists of the present invention are usefulin veterinary and animal husbandry applications to counter weight lossin animals, or to promote growth. For instance, the invention may alsofind use for increasing the efficiency of animal meat production.Specifically, animals may be fed or injected with a hedgehog agonist inorder to increase overall skeletal muscle mass, e.g., to increase theweight of such farm animals as cows, pigs, sheep, chickens and salmon.

The maintenance of tissues and organs ex vivo is also highly desirable.Tissue replacement therapy is well established in the treatment of humandisease. There are many situations where one may wish to transplantmuscle cells, especially muscle stem cells, into a recipient host wherethe recipient's cells are missing, damaged or dysfunctional muscle cellsin muscle wasting disease. For example, transplantation of normalmyoblasts may be useful to treat Duchenne muscular dystrophy and othermuscle degeneration and wasting diseases. See, for example, Partridge(1991) Muscle & Nerve 14:197-212. In the case of myoblasts, they may beinjected at various sites to treat muscle-wasting diseases.

The subject method can be used to regulate the growth of muscle cellsand tissue in vitro, as well as to accelerate the grafting of implantedmuscle tissue to an animal host. In this regard, the present inventionalso concerns myoblast cultures which have been expanded by treatmentwith a hedgehog agonist. In an illustrative embodiment, such a methodcomprises obtaining a muscle sample, preferably one including myoblasts;optionally treating the cell sample enzymically to separate the cells;culturing, in the presence of a hedgehog agonist.

Yet another aspect of the present invention concerns the observation inthe art that ptc, hedgehog, and/or smoothened are involved inmorphogenic signals involved in other vertebrate organogenic pathways inaddition to neuronal differentiation as described above, having apparentroles in other endodermal patterning, as well as both mesodermal andendodermal differentiation processes. Thus, it is contemplated by theinvention that compositions comprising hedgehog agonists can also beutilized for both cell culture and therapeutic methods involvinggeneration and maintenance of non-neuronal tissue.

In one embodiment, the present invention makes use of the discovery thatptc, hedgehog, and smoothened are apparently involved in controlling thedevelopment of stem cells responsible for formation of the digestivetract, liver, lungs, and other organs which derive from the primitivegut. Shh serves as an inductive signal from the endoderm to themesoderm, which is critical to gut morphogenesis. Therefore, forexample, hedgehog agonists of the instant method can be employed forregulating the development and maintenance of an artificial liver whichcan have multiple metabolic functions of a normal liver. In an exemplaryembodiment, the subject method can be used to regulate the proliferationand differentiation of digestive tube stem cells to form hepatocytecultures which can be used to populate extracellular matrices, or whichcan be encapsulated in biocompatible polymers, to form both implantableand extracorporeal artificial livers.

In another embodiment, therapeutic compositions of hedgehog agonists canbe utilized in conjunction with transplantation of such artificiallivers, as well as embryonic liver structures, to regulate uptake ofintraperitoneal implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted liver tissue.

In yet another embodiment, the subject method can be employedtherapeutically to regulate such organs after physical, chemical orpathological insult. For instance, therapeutic compositions comprisinghedgehog agonists can be utilized in liver repair subsequent to apartial hepatectomy.

The generation of the pancreas and small intestine from the embryonicgut depends on intercellular signalling between the endodermal andmesodermal cells of the gut. In particular, the differentiation ofintestinal mesoderm into smooth muscle has been suggested to depend onsignals from adjacent endodermal cells. One candidate mediator ofendodermally derived signals in the embryonic hindgut is Sonic hedgehog.See, for example, Apelqvist et al. (1997) Curr Biol 7:801-4. The Shhgene is expressed throughout the embryonic gut endoderm with theexception of the pancreatic bud endoderm, which instead expresses highlevels of the homeodomain protein Ipf1/Pdx1 (insulin promoter factor1/pancreatic and duodenal homeobox 1), an essential regulator of earlypancreatic development. Apelqvist et al., supra, have examined whetherthe differential expression of Shh in the embryonic gut tube controlsthe differentiation of the surrounding mesoderm into specializedmesoderm derivatives of the small intestine and pancreas. To test this,they used the promoter of the Ipf1/Pdx1 gene to selectively express Shhin the developing pancreatic epithelium. In Ipf1/Pdx1-Shh transgenicmice, the pancreatic mesoderm developed into smooth muscle andinterstitial cells of Cajal, characteristic of the intestine, ratherthan into pancreatic mesenchyme and spleen. Also, pancreatic explantsexposed to Shh underwent a similar program of intestinaldifferentiation. These results provide evidence that the differentialexpression of endodermally derived Shh controls the fate of adjacentmesoderm at different regions of the gut tube.

In the context of the present invention, it is contemplated thereforethat the subject hedgehog agonists can be used to control or regulatethe proliferation and/or differentiation of pancreatic tissue both invivo and in vitro.

There are a wide variety of pathological cell proliferative anddifferentiative conditions for which the agonists of the presentinvention may provide therapeutic benefits, with the general strategybeing, for example, the correction of aberrant insulin expression, ormodulation of differentiation. More generally, however, the presentinvention relates to a method of inducing and/or maintaining adifferentiated state, enhancing survival and/or affecting proliferationof pancreatic cells, by contacting the cells with the subject agonists.For instance, it is contemplated by the invention that, in light of theapparent involvement of ptc, hedgehog, and smoothened in the formationof ordered spatial arrangements of pancreatic tissues, the subjectmethod could be used as part of a technique to generate and/or maintainsuch tissue both in vitro and in vivo. For instance, modulation of thefunction of hedgehog can be employed in both cell culture andtherapeutic methods involving generation and maintenance β-cells andpossibly also for non-pancreatic tissue, such as in controlling thedevelopment and maintenance of tissue from the digestive tract, spleen,lungs, and other organs which derive from the primitive gut.

In an exemplary embodiment, the present method can be used in thetreatment of hyperplastic and neoplastic disorders effecting pancreatictissue, particularly those characterized by aberrant proliferation ofpancreatic cells. For instance, pancreatic cancers are marked byabnormal proliferation of pancreatic cells which can result inalterations of insulin secretory capacity of the pancreas. For instance,certain pancreatic hyperplasias, such as pancreatic carcinomas, canresult in hypoinsulinemia due to dysfunction of β-cells or decreasedislet cell mass. To the extent that aberrant ptc, hedgehog, andsmoothened signaling may be indicated in disease progression, thesubject agonists can be used to enhance regeneration of the tissue afteranti-tumor therapy.

Moreover, manipulation of hedgehog signaling properties at differentpoints may be useful as part of a strategy for reshaping/repairingpancreatic tissue both in vivo and in vitro. In one embodiment, thepresent invention makes use of the apparent involvement of ptc,hedgehog, and smoothened in regulating the development of pancreatictissue. In general, the subject method can be employed therapeuticallyto regulate the pancreas after physical, chemical or pathologicalinsult. In yet another embodiment, the subject method can be applied tocell culture techniques, and in particular, may be employed to enhancethe initial generation of prosthetic pancreatic tissue devices.Manipulation of proliferation and differentiation of pancreatic tissue,for example, by altering hedgehog activity, can provide a means for morecarefully controlling the characteristics of a cultured tissue. In anexemplary embodiment, the subject method can be used to augmentproduction of prosthetic devices which require β-islet cells, such asmay be used in the encapsulation devices described in, for example, theAebischer et al. U.S. Pat. No. 4,892,538, the Aebischer et al. U.S. Pat.No. 5,106,627, the Lim U.S. Pat. No. 4,391,909, and the Sefton U.S. Pat.No. 4,353,888. Early progenitor cells to the pancreatic islets aremultipotential, and apparently coactivate all the islet-specific genesfrom the time they first appear. As development proceeds, expression ofislet-specific hormones, such as insulin, becomes restricted to thepattern of expression characteristic of mature islet cells. Thephenotype of mature islet cells, however, is not stable in culture, asreappearance of embryonic traits in mature β-cells can be observed. Byutilizing the subject hedgehog agonists, the differentiation path orproliferative index of the cells can be regulated.

Furthermore, manipulation of the differentiative state of pancreatictissue can be utilized in conjunction with transplantation of artificialpancreas so as to promote implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted tissue. For instance,manipulation of hedgehog function to affect tissue differentiation canbe utilized as a means of maintaining graft viability.

Bellusci et al. (1997) Development 124:53 report that Sonic hedgehogregulates lung mesenchymal cell proliferation in vivo. Accordingly, thepresent method can be used to regulate regeneration of lung tissue,e.g., in the treatment of emphysema.

In still another embodiment of the present invention, compositionscomprising hedgehog agonists can be used in the in vitro generation ofskeletal tissue, such as from skeletogenic stem cells, as well as the invivo treatment of skeletal tissue deficiencies. The present inventionparticularly contemplates the use of hedgehog agonists to regulate therate of chondrogenesis and/or osteogenesis. By “skeletal tissuedeficiency”, it is meant a deficiency in bone or other skeletalconnective tissue at any site where it is desired to restore the bone orconnective tissue, no matter how the deficiency originated, e.g.,whether as a result of surgical intervention, removal of tumor,ulceration, implant, fracture, or other traumatic or degenerativeconditions.

For instance, the method of the present invention can be used as part ofa regimen for restoring cartilage function to a connective tissue. Suchmethods are useful in, for example, the repair of defects or lesions incartilage tissue which is the result of degenerative wear such as thatwhich results in arthritis, as well as other mechanical derangementswhich may be caused by trauma to the tissue, such as a displacement oftorn meniscus tissue, meniscectomy, a Taxation of a joint by a tornligament, misalignment of joints, bone fracture, or by hereditarydisease. The present reparative method is also useful for remodelingcartilage matrix, such as in plastic or reconstructive surgery, as wellas periodontal surgery. The present method may also be applied toimproving a previous reparative procedure, for example, followingsurgical repair of a meniscus, ligament, or cartilage. Furthermore, itmay prevent the onset or exacerbation of degenerative disease if appliedearly enough after trauma.

In one embodiment of the present invention, the subject method comprisestreating the afflicted connective tissue with a therapeuticallysufficient amount of a hedgehog agonist, particularly an agonistselective for Indian hedgehog signal transduction, to regulate acartilage repair response in the connective tissue by managing the rateof differentiation and/or proliferation of chondrocytes embedded in thetissue. Such connective tissues as articular cartilage, interarticularcartilage (menisci), costal cartilage (connecting the true ribs and thesternum), ligaments, and tendons are particularly amenable to treatmentin reconstructive and/or regenerative therapies using the subjectmethod. As used herein, regenerative therapies include treatment ofdegenerative states which have progressed to the point of whichimpairment of the tissue is obviously manifest, as well as preventivetreatments of tissue where degeneration is in its earliest stages orimminent.

In an illustrative embodiment, the subject method can be used as part ofa therapeutic intervention in the treatment of cartilage of adiarthroidal joint, such as a knee, an ankle, an elbow, a hip, a wrist,a knuckle of either a finger or toe, or a tempomandibular joint. Thetreatment can be directed to the meniscus of the joint, to the articularcartilage of the joint, or both. To further illustrate, the subjectmethod can be used to treat a degenerative disorder of a knee, such aswhich might be the result of traumatic injury (e.g., a sports injury orexcessive wear) or osteoarthritis. The subject agonists may beadministered as an injection into the joint with, for instance, anarthroscopic needle. In some instances, the injected agent can be in theform of a hydrogel or other slow release vehicle described above inorder to permit a more extended and regular contact of the agent withthe treated tissue.

The present invention further contemplates the use of the subject methodin the field of cartilage transplantation and prosthetic devicetherapies. However, problems arise, for instance, because thecharacteristics of cartilage and fibrocartilage varies between differenttissue: such as between articular, meniscal cartilage, ligaments, andtendons, between the two ends of the same ligament or tendon, andbetween the superficial and deep parts of the tissue. The zonalarrangement of these tissues may reflect a gradual change in mechanicalproperties, and failure occurs when implanted tissue, which has notdifferentiated under those conditions, lacks the ability toappropriately respond. For instance, when meniscal cartilage is used torepair anterior cruciate ligaments, the tissue undergoes a metaplasia topure fibrous tissue. By regulating the rate of chondrogenesis, thesubject method can be used to particularly address this problem, byhelping to adaptively control the implanted cells in the new environmentand effectively resemble hypertrophic chondrocytes of an earlierdevelopmental stage of the tissue.

In similar fashion, the subject method can be applied to enhancing boththe generation of prosthetic cartilage devices and to theirimplantation. The need for improved treatment has motivated researchaimed at creating new cartilage that is based oncollagen-glycosaminoglycan templates (Stone et al. (1990) Clin OrthopRelat Red 252:129), isolated chondrocytes (Grande et al. (1989) J OrthopRes 7:208; and Takigawa et al. (1987) Bone Miner 2:449), andchondrocytes attached to natural or synthetic polymers (Walitani et al.(1989) J Bone Jt Surg 71B:74; Vacanti et al. (1991) Plast Reconstr Surg88:753; von Schroeder et al. (1991) J Biomed Mater Res 25:329; Freed etal. (1993) J Biomed Mater Res 27:11; and the Vacanti et al. U.S. Pat.No. 5,041,138). For example, chondrocytes can be grown in culture onbiodegradable, biocompatible highly porous scaffolds formed frompolymers such as polyglycolic acid, polylactic acid, agarose gel, orother polymers which degrade over time as function of hydrolysis of thepolymer backbone into innocuous monomers. The matrices are designed toallow adequate nutrient and gas exchange to the cells until engraftmentoccurs. The cells can be cultured in vitro until adequate cell volumeand density has developed for the cells to be implanted. One advantageof the matrices is that they can be cast or molded into a desired shapeon an individual basis, so that the final product closely resembles thepatient's own ear or nose (by way of example), or flexible matrices canbe used which allow for manipulation at the time of implantation, as ina joint.

In one embodiment of the subject method, the implants are contacted witha hedgehog agonist during certain stages of the culturing process inorder to manage the rate of differentiation of chondrocytes and theformation of hypertrophic chondrocytes in the culture.

In another embodiment, the implanted device is treated with a hedgehogagonist in order to actively remodel the implanted matrix and to make itmore suitable for its intended function. As set forth above with respectto tissue transplants, the artificial transplants suffer from the samedeficiency of not being derived in a setting which is comparable to theactual mechanical environment in which the matrix is implanted. Theability to regulate the chondrocytes in the matrix by the subject methodcan allow the implant to acquire characteristics similar to the tissuefor which it is intended to replace.

In yet another embodiment, the subject method is used to enhanceattachment of prosthetic devices. To illustrate, the subject method canbe used in the implantation of a periodontal prosthesis, wherein thetreatment of the surrounding connective tissue stimulates formation ofperiodontal ligament about the prosthesis.

In still further embodiments, the subject method can be employed as partof a regimen for the generation of bone (osteogenesis) at a site in theanimal where such skeletal tissue is deficient. Indian hedgehog isparticularly associated with the hypertrophic chondrocytes that areultimately replaced by osteoblasts. For instance, administration of ahedgehog agonist of the present invention can be employed as part of amethod for regulating the rate of bone loss in a subject. For example,preparations comprising hedgehog agonists can be employed, for example,to control endochondral ossification in the formation of a “model” forossification.

In yet another embodiment of the present invention, a hedgehog agonistcan be used to regulate spermatogenesis. The hedgehog proteins,particularly Dhh, have been shown to be involved in the differentiationand/or proliferation and maintenance of testicular germ cells. Dhhexpression is initiated in Sertoli cell precursors shortly after theactivation of Sry (testicular determining gene) and persists in thetestis into the adult. Males are viable but infertile, owing to acomplete absence of mature sperm. Examination of the developing testisin different genetic backgrounds suggests that Dhh regulates both earlyand late stages of spermatogenesis. Bitgood et al. (1996) Curr Biol6:298. In a preferred embodiment, a hedgehog agonist can be used as afertility agent. In similar fashion, hedgehog agonists of the subjectmethod are potentially useful for modulating normal ovarian function.

The subject method also has wide applicability to the treatment orprophylaxis of disorders afflicting epithelial tissue, as well as incosmetic uses. In general, the method can be characterized as includinga step of administering to an animal an amount of a hedgehog agonisteffective to alter the growth state of a treated epithelial tissue. Themode of administration and dosage regimens will vary depending on theepithelial tissue(s) which is to be treated. For example, topicalformulations will be preferred where the treated tissue is epidermaltissue, such as dermal or mucosal tissues.

A method which “promotes the healing of a wound” results in the woundhealing more quickly as a result of the treatment than a similar woundheals in the absence of the treatment. “Promotion of wound healing” canalso mean that the method regulates the proliferation and/or growth of,inter alia, keratinocytes, or that the wound heals with less scarring,less wound contraction, less collagen deposition and more superficialsurface area. In certain instances, “promotion of wound healing” canalso mean that certain methods of wound healing have improved successrates, (e.g., the take rates of skin grafts,) when used together withthe method of the present invention.

Complications are a constant risk with wounds that have not fully healedand remain open. Although most wounds heal quickly without treatment,some types of wounds resist healing. Wounds which cover large surfaceareas also remain open for extended periods of time. In one embodimentof the present invention, the subject method can be used to acceleratethe healing of wounds involving epithelial tissues, such as resultingfrom surgery, burns, inflammation or irritation. Certain of the hedgehogagonists of the present invention can also be applied prophylactically,such as in the form of a cosmetic preparation, to enhance tissueregeneration processes, e.g., of the skin, hair and/or fingernails.

Despite significant progress in reconstructive surgical techniques,scarring can be an important obstacle in regaining normal function andappearance of healed skin. This is particularly true when pathologicscarring such as keloids or hypertrophic scars of the hands or facecauses functional disability or physical deformity. In the severestcircumstances, such scarring may precipitate psychosocial distress and alife of economic deprivation. Wound repair includes the stages ofhemostasis, inflammation, proliferation, and remodeling. Theproliferative stage involves multiplication of fibroblasts andendothelial and epithelial cells. Through the use of the subject method,the rate of proliferation of epithelial cells in and proximal to thewound can be controlled in order to accelerate closure of the woundand/or minimize the formation of scar tissue.

Full and partial thickness burns are an example of a wound type whichoften covers large surface areas and therefore requires prolongedperiods of time to heal. As a result, life-threatening complicationssuch as infection and loss of bodily fluids often arise. In addition,healing in burns is often disorderly, resulting in scarring anddisfigurement. In some cases wound contraction due to excessive collagendeposition results in reduced mobility of muscles in the vicinity of thewound. The compositions and method of the present invention can be usedto accelerate the rate of healing of burns and to promote healingprocesses that result in more desirable cosmetic outcomes and less woundcontraction and scarring.

Severe burns which cover large areas are often treated by skinautografts taken from undamaged areas of the patient's body. The subjectmethod can also be used in conjunction with skin grafts to improve“take” rates of the graft by accelerating growth of both the graftedskin and the patient's skin that is proximal to the graft.

Dermal ulcers are yet another example of wounds that are amenable totreatment by the subject method, e.g., to cause healing of the ulcerand/or to prevent the ulcer from becoming a chronic wound. For example,one in seven individuals with diabetes develop dermal ulcers on theirextremities, which are susceptible to infection. Individuals withinfected diabetic ulcers often require hospitalization, intensiveservices, expensive antibiotics, and, in some cases, amputation. Dermalulcers, such as those resulting from venous disease (venous stasisulcers), excessive pressure (decubitus ulcers) and arterial ulcers alsoresist healing. The prior art treatments are generally limited tokeeping the wound protected, free of infection and, in some cases, torestore blood flow by vascular surgery. According to the present method,the afflicted area of skin can be treated by a therapy which includes ahedgehog agonist which promotes epithelization of the wound, e.g.,accelerates the rate of the healing of the skin ulcers.

The present treatment can also be effective as part of a therapeuticregimen for treating oral and paraoral ulcers, e.g., resulting fromradiation and/or chemotherapy. Such ulcers commonly develop within daysafter chemotherapy or radiation therapy. These ulcers usually begin assmall, painful irregularly shaped lesions usually covered by a delicategray necrotic membrane and surrounded by inflammatory tissue. In manyinstances, lack of treatment results in proliferation of tissue aroundthe periphery of the lesion on an inflammatory basis. For instance, theepithelium bordering the ulcer usually demonstrates proliferativeactivity, resulting in loss of continuity of surface epithelium. Theselesions, because of their size and loss of epithelial integrity, lendthe body to potential secondary infection. Routine ingestion of food andwater becomes a very painful event and, if the ulcers proliferatethroughout the alimentary canal, diarrhea usually is evident with allits complicating factors. According to the present invention, atreatment for such ulcers which includes application of a hedgehogagonist can reduce the abnormal proliferation and differentiation of theaffected epithelium, helping to reduce the severity of subsequentinflammatory events.

In another exemplary embodiment, the subject method is provided fortreating or preventing gastrointestinal diseases. Briefly, a widevariety of diseases are associated with disruption of thegastrointestinal epithelium or villi, including chemotherapy- andradiation-therapy-induced enteritis (i.e., gut toxicity) and mucositis,peptic ulcer disease, gastroenteritis and colitis, villus atrophicdisorders, and the like. For example, chemotherapeutic agents andradiation therapy used in bone marrow transplantation and cancer therapyaffect rapidly proliferating cells in both the hematopoietic tissues andsmall intestine, leading to severe and often dose-limiting toxicities.Damage to the small intestine mucosal barrier results in seriouscomplications of bleeding and sepsis. The subject method can be used topromote proliferation of gastrointestinal epithelium and therebyincrease the tolerated doses for radiation and chemotherapy agents.Effective treatment of gastrointestinal diseases may be determined byseveral criteria, including an enteritis score, other tests well knownin the art.

Levine et al. (1997) J Neurosci 17:6277 show that hedgehog proteins canregulate mitogenesis and photoreceptor differentiation in the vertebrateretina, and Ihh is a candidate factor from the pigmented epithelium topromote retinal progenitor proliferation and photoreceptordifferentiation. Likewise, Jensen et al. (1997) Development 124:363demonstrated that treatment of cultures of perinatal mouse retinal cellswith the amino-terminal fragment of Sonic hedgehog protein results in anincrease in the proportion of cells that incorporate bromodeoxuridine,in total cell numbers, and in rod photoreceptors, amacrine cells andMuller glial cells, suggesting that Sonic hedgehog promotes theproliferation of retinal precursor cells. Thus, the subject method canbe used in the treatment of degenerative diseases of retinal cells andregulate photoreceptor differentiation.

With age, the epidermis thins and the skin appendages atrophy. Hairbecomes sparse and sebaceous secretions decrease, with consequentsusceptibility to dryness, chapping, and fissuring. The dermisdiminishes with loss of elastic and collagen fibers Moreover,keratinocyte proliferation (which is indicative of skin thickness andskin proliferative capacity) decreases with age. An increase inkeratinocyte proliferation is believed to counteract skin aging, i.e.,wrinkles, thickness, elasticity and repair. According to the presentinvention, a proliferative form of a hedgehog agonist can be used eithertherapeutically or cosmetically to counteract, at least for a time, theeffects of aging on skin.

Yet another aspect of the present invention relates to the use of thesubject method to promote hair growth. Hair is basically composed ofkeratin, a tough and insoluble protein; its chief strength lies in itsdisulphide bond of cysteine. Each individual hair comprises acylindrical shaft and a root, and is contained in a follicle, aflask-like depression in the skin. The bottom of the follicle contains afinger-like projection termed the papilla, which consists of connectivetissue from which hair grows, and through which blood vessels supply thecells with nourishment. The shaft is the part that extends outwards fromthe skin surface, whilst the root has been described as the buried partof the hair. The base of the root expands into the hair bulb, whichrests upon the papilla. Cells from which the hair is produced grow inthe bulb of the follicle; they are extruded in the form of fibers as thecells proliferate in the follicle. Hair “growth” refers to the formationand elongation of the hair fiber by the dividing cells.

As is well known in the art, the common hair cycle is divided into threestages: anagen, catagen, and telogen. During the active phase (anagen),the epidermal stem cells of the dermal papilla divide rapidly. Daughtercells move upward and differentiate to form the concentric layers of thehair itself. The transitional stage, catagen, is marked by the cessationof mitosis of the stem cells in the follicle. The resting stage is knownas telogen, where the hair is retained within the scalp for severalweeks before an emerging new hair developing below it dislodges thetelogen-phase shaft from its follicle. From this model it has becomeclear that the larger the pool of dividing stem cells that differentiateinto hair cells, the more hair growth occurs. Accordingly, methods forincreasing or reducing hair growth can be carried out by potentiating orinhibiting, respectively, the proliferation of these stem cells.

Thus, in certain embodiments, the subject method can be employed as away of promoting the growth of human hair, e.g., to correct baldness,alopecia, or other diseases characterized by hair loss.

The subject method can also be used in treatment of a wound to eyetissue. Generally, damage to corneal tissue, whether by disease, surgeryor injury, may affect epithelial and/or endothelial cells, depending onthe nature of the wound. Corneal epithelial cells are thenon-keratinized epithelial cells lining the external surface of thecornea and provide a protective barrier against the externalenvironment. Corneal wound healing has been of concern to bothclinicians and researchers. Ophthalmologists are frequently confrontedwith corneal dystrophies and problematic injuries that result inpersistent and recurrent epithelial erosion, often leading to permanentendothelial loss. The use of proliferative forms of the subject hedgehogagonists can be used in these instances to promote epithelialization ofthe affected corneal tissue.

To further illustrate, specific disorders typically associated withepithelial cell damage in the eye, and for which the subject method canprovide beneficial treatment, include persistent corneal epithelialdefects, recurrent erosions, neurotrophic corneal ulcers,keratoconjunctivitis sicca, microbial corneal ulcers, viral corneaulcers, and the like. Surgical procedures typically causing injury tothe epithelial cell layers include laser procedures performed on theocular surface, any refractive surgical procedures such as radialkeratotomy and astigmatic keratotomy, conjunctival flaps, conjunctivaltransplants, epikeratoplasty, and corneal scraping. Moreover,superficial wounds such as scrapes, surface erosion, inflammation, etc.can cause lose of epithelial cells. According to the present invention,the corneal epithelium is contacted with an amount of a hedgehog agonisteffective to cause proliferation of the corneal epithelial cells toappropriately heal the wound.

In another aspect of the invention, the subject method can be used toinduce differentiation and/or promote proliferation of epitheliallyderived tissue. Such forms of these molecules can provide a basis fordifferentiation therapy for the treatment of hyperplastic and/orneoplastic conditions involving epithelial tissue. For example, suchpreparations can be used for the treatment of cutaneous diseases inwhich there is abnormal proliferation or growth of cells of the skin.

The present method can be used for improving the “take rate” of a skingraft. Grafts of epidermal tissue can, if the take rate of the graft isto long, blister and shear, decreasing the likelihood that the autograftwill “take”, i.e. adhere to the wound and form a basement membrane withthe underlying granulation tissue. Take rates can be increased by thesubject method by inducing proliferation of the keratinocytes. Themethod of increasing take rates comprises contacting the skin autograftwith an effective wound healing amount of a hedgehog agonist describedin the method of promoting wound healing and in the method of promotingthe growth and proliferation of keratinocytes, as described above.

Skin equivalents have many uses not only as a replacement for human oranimal skin for skin grafting, but also as test skin for determining theeffects of pharmaceutical substances and cosmetics on skin. A majordifficulty in pharmacological, chemical and cosmetic testing is thedifficulties in determining the efficacy and safety of the products onskin. One advantage of the skin equivalents of the invention is theiruse as an indicator of the effects produced by such substances throughin vitro testing on test skin.

Thus, in one embodiment of the subject method can be used as part of aprotocol for skin grafting of, e.g., denuded areas, granulating woundsand burns. The use of hedgehog agonists can enhance such graftingtechniques as split thickness autografts and epidermal autografts(cultured autogenic keratinocytes) and epidermal allografts (culturedallogenic keratinocytes). In the instance of the allograft, the use ofthe subject method to enhance the formation of skin equivalents inculture helps to provide/maintain a ready supply of such grafts (e.g.,in tissue banks) so that the patients might be covered in a singleprocedure with a material which allows permanent healing to occur.

In this regard, the present invention also concerns composite livingskin equivalents comprising an epidermal layer of cultured keratinocytecells which have been expanded by treatment with a hedgehog agonist. Thesubject method can be used as part of a process for the preparation ofcomposite living skin equivalents. In an illustrative embodiment, such amethod comprises obtaining a skin sample, treating the skin sampleenzymically to separate the epidermis from the dermis, treating theepidermis enzymically to release the keratinocyte cells, culturing, inthe presence of a hedgehog agonist, the epidermal keratinocytes untilconfluence, in parallel, or separately, treating the dermisenzymatically to release the fibroblast cells, culturing the fibroblastscells until sub-confluence, inoculating a porous, cross-linked collagensponge membrane with the cultured fibroblast cells, incubating theinoculated collagen sponge on its surface to allow the growth of thefibroblast cells throughout the collagen sponge, and then inoculating itwith cultured keratinocyte cells, and further incubating the compositeskin equivalent complex in the presence of a hedgehog agonist to promotethe growth of the cells.

In other embodiments, skin sheets containing both epithelial andmesenchymal layers can be isolated in culture and expanded with culturemedia supplemented with a proliferative form of a hedgehog agonist. Anyskin sample amenable to cell culture techniques can be used inaccordance with the present invention. The skin samples may be autogenicor allogenic.

In another aspect of the invention, the subject method can be used inconjunction with various periodontal procedures in which control ofepithelial cell proliferation in and around periodontal tissue isdesired. In one embodiment, hedgehog agonists can be used to enhancereepithelialization around natural and prosthetic teeth, e.g., topromote formation of gum tissue.

Hedgehog gene products are able to regulate maturation of T lymphocytes.Certain aspects of the invention are directed to hedgehog agonists andtheir uses as immunomodulatory agents against both acquired andhereditary immunological disorders.

For instance, such compositions can be used to increase the populationof T-helper cells to optimum levels in the host, e.g., to stimulate theimmune system of the animal. Such uses of the subject compositions canbe used in the treatment of bacterial or viral infections, as well as tohelp the body fight against cancer cells. Alternatively, thesesubstances also enable the host to adjust to diseases arising fromdisarrangement of self-recognition processes in which there is excessiveattack by host T-cells against endogenous tissues. In such instances,the subject compositions can be used to reduce T-cell population so thatthe signs and symptoms of self-directed inflammatory (autoimmune)diseases such rheumatoid arthritis and multiple sclerosis areameliorated.

As described herein, hedgehog proteins inhibit maturation of Tlymphocytes. Based upon its inhibitory effect, the administration ofhedgehog agonists is suggested herein as a treatment for several typesof immunological disorders involving unwanted activation of cellularimmunity, e.g., graft rejection, autoimmune disorders, and the like.

In general, the method of the present invention comprises administeringto animal, or to cultured lymphocytes in vitro, an amount of a hedgehogagonist which produces a non-toxic response by the cell of inhibition ofmaturation. The subject method can be carried out on cells which may beeither dispersed in culture or a part of an intact tissue or organ.Moreover, the method can be performed on cells which are provided inculture (in vitro), or on cells in a whole animal (in vivo). Theinvention also relates to methods of controlling the functionalperformance of T cells by use of the pharmaceutical preparations of theinvention.

Without wishing to be bound by any particular theory, the inhibitoryeffect of hedgehog on T cell maturation may be due at least in part tothe ability of hedgehog proteins to antagonize (directly or indirectly)patched-mediated regulation of gene expression and other physiologicaleffects mediated by that protein. The patched gene product, a cellsurface protein, is understood to signal through a pathway which causestranscriptional repression of members of the Wnt and Dpp/BMP families ofmorphogens, proteins which impart positional information. In othertissue, the introduction of hedgehog relieves (derepresses) thisinhibition conferred by patched, allowing expression of particular geneprograms.

In another aspect, the present invention provides pharmaceuticalpreparations comprising hedgehog agonists. The hedgehog agonists for usein the subject method may be conveniently formulated for administrationwith a biologically acceptable medium, such as water, buffered saline,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol and the like) or suitable mixtures thereof. The optimumconcentration of the active ingredient(s) in the chosen medium can bedetermined empirically, according to procedures well known to medicinalchemists. As used herein, “biologically acceptable medium” includes anyand all solvents, dispersion media, and the like which may beappropriate for the desired route of administration of thepharmaceutical preparation. The use of such media for pharmaceuticallyactive substances is known in the art. Except insofar as anyconventional media or agent is incompatible with the activity of thehedgehog agonist, its use in the pharmaceutical preparation of theinvention is contemplated. Suitable vehicles and their formulationinclusive of other proteins are described, for example, in the bookRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences. Mack Publishing Company, Easton, Pa., USA 1985). Thesevehicles include injectable “deposit formulations”.

Pharmaceutical formulations of the present invention can also includeveterinary compositions, e.g., pharmaceutical preparations of thehedgehog agonists suitable for veterinary uses, e.g., for the treatmentof livestock or domestic animals, e.g., dogs.

Rechargeable or biodegradable devices may also provide methods ofintroduction. Various slow release polymeric devices have been developedand tested in vivo in recent years for the controlled delivery of drugs,including proteinaceous biopharmaceuticals. A variety of biocompatiblepolymers (including hydrogels), including both biodegradable andnon-degradable polymers, can be used to form an implant for thesustained release of a hedgehog agonist at a particular target site.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, controlled release patch, etc.,administration by injection, infusion or inhalation; topical by lotionor ointment; and rectal by suppositories. Oral and topicaladministrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms such as described below orby other conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular hedgehog agonist employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous,intracerebroventricular, and subcutaneous doses of the compounds of thisinvention for a patient will range from about 0.0001 to about 100 mg perkilogram of body weight per day, preferably from about 0.001 to about 10mg per kilogram, even more preferably from about 0.01 to about 1 mg perkilogram.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The term “treatment” is intended to encompass also prophylaxis, therapy,and cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable and/or sterile carriers andcan also be administered in conjunction with other antimicrobial agentssuch as penicillins, cephalosporins, aminoglycosides, and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticeffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

V. Pharmaceutical Compositions

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The hedgehog agonistsaccording to the invention may be formulated for administration in anyconvenient way for use in human or veterinary medicine. In certainembodiments, the compound included in the pharmaceutical preparation maybe active itself, or may be a prodrug, e.g., capable of being convertedto an active compound in a physiological setting.

Thus, another aspect of the present invention provides pharmaceuticallyacceptable compositions comprising a therapeutically effective amount ofone or more of the compounds described above, formulated together withone or more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail below, the pharmaceutical compositionsof the present invention may be specially formulated for administrationin solid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular or intravenous injection as, forexample, a sterile solution or suspension; (3) topical application, forexample, as a cream, ointment or spray applied to the skin; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam. However, in certain embodiments the subject compounds may besimply dissolved or suspended in sterile water. In certain embodiments,the pharmaceutical preparation is non-pyrogenic, i.e., does not elevatethe body temperature of a patient.

The phrase “therapeutically effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animaland thereby blocking the biological consequences of that pathway in thetreated cells, at a reasonable benefit/risk ratio applicable to anymedical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agonists fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present hedgehog agonistsmay contain a basic functional group, such as amino or alkylamino, andare, thus, capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred per cent, this amount will range fromabout 1 per cent to about ninety-nine percent of active ingredient,preferably from about 5 per cent to about 70 per cent, most preferablyfrom about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar—agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in microencapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar—agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active hedgehog agonist.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the hedgehog agonists inthe proper medium. Absorption enhancers can also be used to increase theflux of the hedgehog agonists across the skin. The rate of such flux canbe controlled by either providing a rate controlling membrane ordispersing the compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”, W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feeds andFeeding” O and B books, Corvallis, Ore., U.S.A., 1977).

VI. Synthetic Schemes and Identification of Active Agonists

The subject inhibitors, and congeners thereof, can be prepared readilyby employing the cross-coupling technologies of Suzuki, Stille, and thelike. These coupling reactions are carried out under relatively mildconditions and tolerate a wide range of “spectator” functionality.

a. Combinatorial Libraries

The compounds of the present invention, particularly libraries ofvariants having various representative classes of substituents, areamenable to combinatorial chemistry and other parallel synthesis schemes(see, for example, PCT WO 94/08051). The result is that large librariesof related compounds, e.g., a variegated library of compoundsrepresented above, can be screened rapidly in high throughput assays inorder to identify potential hedgehog agonist lead compounds, as well asto refine the specificity, toxicity, and/or cytotoxic-kinetic profile ofa lead compound. For instance, ptc, hedgehog, or smoothened bioactivityassays can be used to screen a library of the subject compounds forthose having antagonist activity toward ptc or agonist activity towardshedgehog or smoothened.

Simply for illustration, a combinatorial library for the purposes of thepresent invention is a mixture of chemically related compounds which maybe screened together for a desired property. The preparation of manyrelated compounds in a single reaction greatly reduces and simplifiesthe number of screening processes which need to be carried out.Screening for the appropriate physical properties can be done byconventional methods.

Diversity in the library can be created at a variety of differentlevels. For instance, the substrate aryl groups used in thecombinatorial reactions can be diverse in terms of the core aryl moiety,e.g., a variegation in terms of the ring structure, and/or can be variedwith respect to the other substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjecthedgehog agonists. See, for example, Blondelle et al. (1995) TrendsAnal. Chem. 14:83; the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899:the Ellman U.S. Pat. No. 5,288,514: the Still et al. PCT publication WO94/08051; the ArQule U.S. Pat. Nos. 5,736,412 and 5,712,171; Chen et al.(1994) JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT publicationsWO92/10092, WO93/09668 and WO91/07087; and the Lerner et al. PCTpublication WO93/20242). Accordingly, a variety of libraries on theorder of about 100 to 1,000,000 or more diversomers of the subjecthedgehog agonists can be synthesized and screened for particularactivity or property.

In an exemplary embodiment, a library of candidate hedgehog agonistsdiversomers can be synthesized utilizing a scheme adapted to thetechniques described in the Still et al. PCT publication WO 94/08051,e.g., being linked to a polymer bead by a hydrolyzable or photolyzablegroup, optionally located at one of the positions of the candidateagonists or a substituent of a synthetic intermediate. According to theStill et al. technique, the library is synthesized on a set of beads,each bead including a set of tags identifying the particular diversomeron that bead. The bead library can then be “plated” withhedgehog-responsive cells. The diversomers can be released from thebead, e.g., by hydrolysis.

The structures of the compounds useful in the present invention lendthemselves readily to efficient synthesis. The nature of the structuresof the subject compounds, as generally set forth above, allows the rapidcombinatorial assembly of such compounds. For example, as in the schemeset forth below, an activated aryl group, such as an aryl triflate orbromide, attached to a bead or other solid support can be linked toanother aryl group by performing a Stille or Suzuki coupling with anaryl stannane or an aryl boronic acid. If the second aryl group isfunctionalized with an aldehyde, an amine substituent can be addedthrough a reductive amination. Alternatively, the second aryl groupcould be functionalized with a leaving group, such as a triflate,tosylate, or halide, capable of being displaced by an amine. Or, thesecond aryl group may be functionalized with an amine group capable ofundergoing reductive amination with an amine, e.g., CyKNH₂. Otherpossible coupling techniques include transition metal-mediated aminearylation reactions. The resultant secondary amine can then be fartherfunctionalized by an acylation, alkylation, or arylation to generate atertiary amine or amide which can then be cleaved from the resin orsupport. These reactions generally are quite mild and have beensuccessfully applied in combinatorial solid-phase synthesis schemes.Furthermore, the wide range of substrates and coupling partners suitableand available for these reactions permits the rapid assembly of large,diverse libraries of compounds for testing in assays as set forthherein. For certain schemes, and for certain substitutions on thevarious substituents of the subject compounds, one of skill in the artwill recognize the need for masking certain functional groups with asuitable protecting group. Such techniques are well known in the art andare easily applied to combinatorial synthesis schemes.

Many variations on the above and related pathways permit the synthesisof widely diverse libraries of compounds which may be tested as agonistsof hedgehog function.

b. Screening Assays

There are a variety of assays available for determining the ability of acompound to antagonize ptc function or agonize smoothened or hedgehogfunction, many of which can be disposed in high-throughput formats. Inmany drug-screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Thus, libraries of synthetic and natural products can be sampled forother compounds which are hedgehog agonists.

In addition to cell-free assays, test compounds can also be tested incell-based assays. In one embodiment, cell which are responsive to theaddition of hedgehog protein can be contacted with a test agent ofinterest, with the assay scoring for, e.g., promotion of proliferationof the cell in the presence of the test agent.

A number of gene products have been implicated in patched-mediatedsignal transduction, including patched, transcription factors of thecubitus interruptus (ci) family, the serine/threonine kinase fused (fu)and the gene products of costal-2, smoothened and suppressor of fused.

The induction of cells by hedgehog proteins sets in motion a cascadeinvolving the activation and inhibition of downstream effectors, theultimate consequence of which is, in some instances, a detectable changein the transcription or translation of a gene. Potential transcriptionaltargets of hedgehog-mediated signaling are the patched gene (Hidalgo andIngham, 1990 Development 110, 291-301; Marigo et al., 1996 ) and thevertebrate homologs of the drosophila cubitus interruptus gene, the Gligenes (Hui et al. (1994) Dev Biol 162:402-413). Patched gene expressionhas been shown to be induced in cells of the limb bud and the neuralplate that are responsive to Shh. (Marigo et al. (1996) PNAS 93:9346-51;Marigo et al. (1996) Development 122:1225-1233). The Gli genes encodeputative transcription factors having zinc finger DNA binding domains(Orenic et al. (1990) Genes & Dev 4:1053-1067; Kinzler et al. (1990) MolCell Biol 10:634-642). Transcription of the Gli gene has been reportedto be upregulated in response to hedgehog in limb buds, whiletranscription of the Gli3 gene is downregulated in response to hedgehoginduction (Marigo et al. (1996) Development 122:1225-1233). By selectingtranscriptional regulatory sequences from such target genes, e.g., frompatched or Gli genes, that are responsible for the up- ordown-regulation of these genes in response to hedgehog signalling, andoperatively linking such promoters to a reporter gene, one can derive atranscription based assay which is sensitive to the ability of aspecific test compound to modify hedgehog-mediated signalling pathways.Expression of the reporter gene, thus, provides a valuable screeningtool for the development of compounds that act as agonists of hedgehog.

Reporter gene based assays of this invention measure the end stage ofthe above-described cascade of events, e.g., transcriptional modulation.Accordingly, in practicing one embodiment of the assay, a reporter geneconstruct is inserted into the reagent cell in order to generate adetection signal dependent on activation of the hedgehog pathway, orstimulation by Shh itself. The amount of transcription from the reportergene may be measured using any method known to those of skill in the artto be suitable. For example, mRNA expression from the reporter gene maybe detected using RNAse protection or RNA-based PCR, or the proteinproduct of the reporter gene may be identified by a characteristic stainor an intrinsic biological activity. The amount of expression from thereporter gene is then compared to the amount of expression in either thesame cell in the absence of the test compound or it may be compared withthe amount of transcription in a substantially identical cell that lacksthe target receptor protein. Any statistically or otherwise significantincrease in the amount of transcription indicates that the test compoundhas in some manner antagonized the normal ptc signal (or agonized thehedgehog or smoothened signal), e.g., the test compound is a potentialhedgehog agonist.

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

In the experimental section below, the term ‘Hh protein’ is used todesignate octyl-Shh-N, a lipophilic form of a bacterially derivedfragment of human sonic hedgehog protein (amino acids 24-198, Shh-N).Specifically, Shh-N has been covalently linked in vitro via its aminoterminal cysteine to an octyl maleimide group. This modified form, likeothers described recently (Pepinsky et al., J. Biol. Chem. 1998, 273,14037-45) exhibits higher specific potency than the correspondingunmodified fragment in several cell-based assays of hedgehog signalling.

Compound Screening

To measure the hedgehog agonist activity of compounds, we used[10T1/2(s12)] cells containing the hedgehog-responsive Gli-Lucreporter-construct. In each MTP (MicroTiter Plate; 96-well plate),10,000-20,000 cells were plated per well, in full media (10% FBS). Afterabout 24-48 hr, plates were switched to low-serum media (=0.5% FBS).Subsequently, a test compound was added at 1-5 μM in the presence orabsence of octyl-hedgehog (see below). After another 24 hr, the mediafrom the MTPs was discarded and replaced with luciferase assay-mix,containing lysis-buffer with luciferase substrate. The plates wereincubated at RT for about 15-30 min and read in a luminometer.

Screen A

Plates were incubated for 48 hr before switching to low-serum. Compoundswere screened at 1-2 μM in the presence of Hh protein (0.01 μg/ml;EC₃₀=about 30% of max-induced activity).

Screen B

Plates were incubated for 24 hr before switching to low-serum. Compoundswere screened at 5 μM without adding Hh protein.

Compound Counter-screen (SV-Luc)

For the counter-screen we used the [10T1/2(SV-Luc)] cells containing aSV40-luciferase expression cassette that allows for a constitutive levelof luciferase-activity in the cells. This assay allows one to assess thespecificity of the compounds selected in the Gli-Luc assay, i.e.,whether the compounds specifically stimulate the hedgehog signallingpathway only, or reporter-constructs in general. Cell plating andcultivation as well as compound handling were performed as in theGli-Luc assay. In this assay, no Hh protein was added because thereporter-construct is constitutively active already.

From Screen A, we identified hits identified in FIGS. 32 and 33. The1,4-diaminocyclohexane subunit of the compounds of FIG. 32 which includethis moiety have the two amino substituents disposed in atrans-relationship, e.g., both substituents equatorial on thecyclohexane subunit. These hits were confirmed in Screen B. The datashown in FIGS. 34a and 34 b essentially involved dosing the twocompounds in both assays (Gli-Luc & SV-Luc).

The data from the Gli-Luc assay is converted to percent activity wherefull activity with Hh protein is set at 100% (FIG. 35; Table 1). Thedata shows that the two compounds can significantly stimulate activityfrom the Gli-luc reporter construct (i.e., activate hedgehog signalling)even in the absence of Hh protein; concentration range 0.1-15 μM, EC50=2μM, EC_(max)=50-70% of max-induced activity with Hh protein).Furthermore, the compounds show a more striking induction in thepresence of hedgehog (EC₅₀=0.2-0.3 μM, EC_(max)=70-90% of max inducedactivity with Hh protein), indicating a synergy between the compoundsand Hh protein, consistent with the compounds serving as agonists forthe hedgehog pathway.

TABLE 1 Compound EC50 (μM) Compound EC50 (μM) A  A′ <10 B <10  B′ <10 C<1  C′ <10 D <0.1  D′ <10 E <10  E′ <10 F <10  F′ <10 G <10  G′ <10 H <1 H′ >10 I >10  I′ <1 J <1  J′ <1 K <1  K′ <10 L <0.05  L′ <0.05 M <1  M′<10 N <1  N′ <1 O <1  O′ <1 P <1  P′ <1 Q <1  Q′ <1 R <1  R′ <0.05 S <1 S′ <0.1 T <10  T′ <0.5 U <10  U′ <1 V <0  V′ <1 W <10  W′ <10 X <10  X′<10 Y <10  Y′ <10 Z <10  Z′ <10  A″ <10  B″ <10  C″ <10  D″ <10  E″ <10 F″ <0.1  G″ <0.1  H″ <0.1  I″ <0.5  J″ <0.5  K″ <0.5  L″ <1  M″ <1  N″<1  O″ <1  P″ <10  Q″ <1  R″ <1  S″ <10  T″ <10  U″ <0.005  V″ <0.005 W″ <0.005  X″ <0.005  Y″ <0.05  Z″ <0.05  A′′′ <0.05  B′′′ <0.05  C′′′<0.05  D′′′ <0.05  E′′′ <0.05  F′′′ <0.05  G′′′ <5  H′′′ <0.05  I′′′<0.05  J′′′ <0.5  K′′′ <5  L′′′ <5  M′′′ <0.5  N′′′ <5  O′′′ <1  P′′′ <5 Q′′′ <1  R′′′ <1  S′′′ <1  T′′′ <1  U′′′ <1

The data from the SV-Luc assay (FIG. 35) indicate that the two compoundsdo not affect the luciferase activity over the range of concentrations(up to 15 μM) tested, suggesting that the compounds do not stimulateactivity from a reporter-construct per se, but are probably specific tothe hedgehog pathway. Also, the unaffected activity suggests that thecompounds are not intrinsically toxic to the cells.

Hh Agonist: Quantitative RT-PCR Measurement of Gli Upregulation

The compounds of FIGS. 32 and 33 were tested for their ability toactivate transcription of two well-studied targets of the Hedgehogpathway: the transcription factor Gli-1, and the putative Hedgehogreceptor component Ptc-1. As depicted in FIG. 35, we found that, at acompound concentration of 8 μM, induction of both targets wasapproximately 60% for the p-cyanophenyl compound B and 40% for them-nitrophenyl compound A of that obtained with the optimal concentrationof Hh protein.

Assays were performed as follows:

Murine C3H 10T1/2 cells were seeded at approximately 200,000 cells perwell in a 24-well plate in complete medium (10% FBS). After 24 hr,medium was removed and replaced with “starvation” medium (0.5% FBS)containing dilutions of compound or Hh protein. After 16-18 hr, totalRNA was prepared using TriZol (Life Technologies, inc.).

One-microgram aliquots of total RNA were used to prepare randomhexamer-primed cDNA with M-MTV Reverse transcriptase (Life Technologies,Inc.).

To measure the relative levels of Gli-1 and Ptc-1 transcripts, TaqManassays (PE Biosystems) were performed with an ABI Prism 7700 SequenceDetection System. As an internal control, GAPDH transcript levels weresimultaneously measured in each reaction. Data were analyzed usingSequence Detector v1.6.3 (Perkin Elmer).

Induction of Cerebellar Neuron Precursor Proliferation by HedgehogSignaling Agonists

In order to test the efficacy of Hedgehog signaling agonists, theability of the agonists to stimulate the proliferation of cerebellarneuron precursors was determined. Cerebellar granule neuron precursorsare known to respond to Hh protein by proliferating. To test theagonists, cerebellar neurons were dissected out of postnatal (one week)rat brains, and placed into primary cell culture. Treatment agents wereadded once, on the first day of culture (0 DIV). Cells were left inculture until 2 DIV, when 3H-thymidine was added for 5 hours; the amountof incorporation by the cells of this 3H-thymidine provides a measure ofthe level of proliferation of the cells. Cells were then lysed, and theincorporation of 3H-thymidine was determined. The positive control wasHh protein, at 1.0 μg/ml final concentration. Hh protein alone causedapproximately a 20-fold increase in 3H-thymidine incorporation,consistent with the known ability of Shh to stimulate proliferation ofthese cells. Non-Hh-protein-stimulated cells, including cells treatedwith control vehicle (DMSO), had very low levels of proliferation.However, the two of the Hh agonists in FIG. 32 that were tested, A andB, were found to significantly stimulate 3H-thymidine incorporation inthe absence of Hh protein treatment, as depicted in FIG. 36. At 5.0 μM,the p-cyanophenyl compound B caused 10.2-fold induction of 3H-thymidineincorporation. FIG. 37 shows that agonist D also strongly stimulated3H-thymidine incorporation in the absence of Shh. At 1.0, 0.3, or 0.1μM, D caused 16-, 17-, or 13-fold induction, respectively, of 3Hthymidine incorporation into the cells. These observations demonstratethat the hedgehog agonists can stimulate known hedgehog biologicalresponses in target cells.

Nerve Crush Assay

CD-1 male mice were anesthetized with Avertin, 240 mg/kg IP. The area onthe ventral side of the mouse leg around the knee was shaved and cleanedwith 70% ethanol to remove the hair followed by swabbing with Betadine.The skin over the thigh was tented up with forceps and small scissorswere used to make a ¼-inch cut in the skin. Fat was moved away to exposethe femoral nerve, artery, and vein. With the tips of the curved #7forceps pointing down, the muscle was spread apart just below thefemoral artery/vein to reveal the sciatic nerve deep in the muscle.While using one pair of forceps to hold the muscle apart, a second pairwas used to carefully lift up the nerve. Care was taken to not lift upthe muscle fibers. The forceps were opened and closed 3 times toseparate the nerve from the muscle. The nerve was held elevated from themuscle with the curved forceps and the hemostats were clamped down onthe nerve to keep the nerve in the middle of the hemostats. Thehemostats were held in place for 10 seconds. In this manner, bothsciatic nerves were crushed approximately 1+ cm above the knee (andbranches). The hemostats were unclipped and the nerve fell back into themuscle. A small pipette tip (P2) was used to apply a small amount ofhistological tissue marking dye to the crushed area. A surgical clip wasused to close the incision. Care was taken not to clip the skin to themuscle. The clips were left in place throughout the entire experiment.

Control surgery was done to a group of mice. This involved lifting thenerve up with the forceps and letting it fall back into place withoutany crush. This site can also be marked with the histological dye.

Drug treatment began on the day of surgery. For Shh protein, a fusionprotein of Shh and immunoglobin (as described in U.S. ProvisionalApplication No. 60/164025, incorporated herein by reference)administered at a dose of 1 mg/kg in a solution of PBS, a subcutaneousinjection was given in the middle of the back of the animal with a 28gauge, ½ cc insulin syringe and repeated every other day until day12-13. (Recovery was complete by then.) For agonist administration, asolution of the agonist in 43% DMSO/PBS (FIGS. 38A and 39B), or in 10%DMSO/water (FIGS. 38B and 39D), was delivered by a minipump (1 μL/hour).Behavioral tests were initiated on day 4 post-surgery and continueduntil day 12 or 13.

Behavioral Testing—Grip Assay

A mouse was placed on an 8″×8″ metal wire grid (like a test-tube rack)with 1 cm openings, and the grid was slowly inverted 10 times with aconstant steady motion. The number of times the mouse failed to gripwith its left and right hind limbs was recorded. The mouse was kept awayfrom the edges of the grid by repositioning the mouse on the grid asnecessary. If the mouse just hooked its leg around or through the gridit was considered a failure. An inversion was repeated if the mouse waswalking, and failed to grip the grid.

Failures of the left and right leg were pooled with other animals fromthat experimental group (6 animals/group×2 foot grip scores/animal=12grip scores/group on any given time point). Results for agonist D aredepicted in FIG. 38A. Animals treated with vehicle alone generally beganto recover on their own by day 9 post-surgery. FIG. 38B depicts resultsobtained using agonist D dissolved in a vehicle of 10% DMSO/water in thetreatment protocol.

Behavioral Testing—Toespread Measurements

Toespread measuring began at Day 4 post-surgery and was measured everyother day. The mouse was held by the proximal part of the tail andpermitted to hold onto the wire top of the cage with its front limbs. Asmall paintbrush or cotton applicator was used to paint the hind toesand footpads of the mouse. The mouse was allowed to walk across a cleansheet of paper, to leave at least two clean prints for each hind limb.Using a ruler, a line was drawn through the widest toe prints on eachfoot and the distance between them was measured. As the animalsrecovered, the distance increased to normal measurements.

The left and right leg scores were averaged and pooled with the otheranimals from that experimental group (6 animals/group×2 foot gripscores/animal=12 toe spread scores/group on any given time point).Results using agonist D are depicted in FIGS. 39A and B. FIG. 39Ddepicts the effects of agonist D dissolved in a vehicle of 10%DMSO/water in this protocol.

Reporter Mice Assays

Mice with a β-galactosidase transgene (ptc-lacZ mice) under theregulatory control of the patched locus express the β-galactosidaseprotein in the same cells where the endogenous mouse patched gene isexpressed. The β-galactosidase protein can thus be used as a faithfulreporter of the expression of the endogenous patched gene. The patchedgene is a known component of the hedgehog signalling pathway and isupregulated when the hedgehog pathway is activated. Hence, in theptc-lacZ mice β-galactosidase protein is overexpressed in mice in whichthe hedgehog pathway has been activated, resulting in more intense bluestaining (due to higher levels of the β-galactosidase enzyme) whentissues are stained for enzymatic activity with the X-gal substrate.

Ptc-lacZ mice were divided into four treatment groups and were treatedwith the following compounds or combinations of compounds for four days,beginning on the first day after birth (dipal-Shh=dipalmitoylated Sonichedgehog):

1) dipal-Shh 10 mg/kg/injection 2X/day 2) dipal-Shh  1 mg/kg/injection2X/day vehicle 10-15 μl/injection 4X/day 3) dipal-Shh  1 mg/kg/injection2X/day agonist B 15 mg/kg/injection 4X/day 4) no treatment

The vehicle for the agonist was 10% DMSO in PBS (pH 7.2). The agonistwas injected from a 4.67 mM stock dissolved in the vehicle solution.

18 hours following the last injections, the mice were sacrificed and thefollowing tissues were collected: skull, kidney, lung, scapula, skin andheart. All tissues were fixed in 0.2% glutaraldehyde, 5 mM EDTA (pH8.0), 20 mM MgCl₂, 100 mM Na₂HPO₄ for 30 minutes before staining in 1mg/ml X-gal, 12.5 mM potassium ferrocyanide, 12.5 mM potassiumferricyanide, 2 mM MgCl₂, 0.01% deoxycholate, 0.02% NP-40, and 100 mMNa₂HPO₄ for 30 hours. Tissues were judged for relative intensity andphotographed.

All tissues from treatment group 3 (agonist+low dose dipal-Shh) showedsignificant upregulation of β-galactosidase (as visualized by moreintense blue staining) compared to those from treatment group 2(vehicle+low dose dipal-Shh), as seen in FIG. 40, wherein tissues fromgroup 3 are depicted on the right side of each frame. The images in FIG.41 show an example of tissue from each of the four treatment groups. Theupregulation observed in group 3 was similar to that seen in treatmentgroup 1 (high dose dipal-Shh). The level of β-galactosidase staining intreatment group 2 was similar to that seen in treatment group 4 (notreatment).

Agonist B was clearly capable of upregulating the hedgehog pathway invivo in the presence of a low dose of dipal-shh which by itself isinsufficient to produce detectable upregulation.

FIG. 42: Ptc-lacZ mice were divided into two treatment groups and weretreated with the following compounds for three days, beginning on thefirst day after birth:

1) vehicle 8-15 μl/injection 4X/day 2) agonist D 4 mg/kg/injection4X/day

The vehicle for the agonist was 10% DMSO in PBS (pH 7.2). The agonistwas injected from a 1.0 mM stock dissolved in the vehicle solution.

Eighteen hours following the last injections, the mice were sacrificedand the forelimbs were collected and processed as described above. Theforelimbs from the agonist-treated mice showed strong upregulation inthe nerves, blood vessels, cartilage, and connective tissue.

Agonist D was clearly capable of upregulating the hedgehog pathway invivo in the absence of dipal-Shh. The upregulation seen at this dose ofagonist D is greater than that seen with injections of dipal-Shh at 10mg/kg/injection, 2×/day.

In a third experiment, Ptc-lacZ mice were divided into five treatmentgroups and were treated with the following compounds for four days,beginning on the first day after birth:

1) vehicle 9-20 μl/injection 4X/day 2) agonist D 0.9 mg/kg/injection4X/day 3) agonist D 0.3 mg/kg/injection 4X/day 4) agonist D 0.1mg/kg/injection 4X/day 5) octyl-shh  10 mg/kg/injection 2X/day

The vehicle for the agonist was 10% DMSO in PBS (pH 7.2). The agonistwas injected from stocks of 0.3, 0.1 and 0.03 mM dissolved in thevehicle solution.

Eighteen hours following the last injections, the mice were sacrificedand the forelimbs were collected and processed as described above.Results of this experiment are depicted in FIG. 43, in which the vehiclecontrol is not shown. The forelimbs from the agonist-treated mice againshowed strong upregulation in the nerves, blood vessels, cartilage, andconnective tissue (compared to the vehicle group). The 0.9 mg/kg groupshowed the highest levels of upregulation in this experiment. Both the0.9 mg/kg and the 0.3 mg/kg dose groups showed greater upregulation thanthe 10 mg/kg Hh protein group. The 0.1 mg/kg group showed very weakupregulation that was clearly less than that seen in the Hh proteingroup.

Agonist D was clearly capable of upregulating the hedgehog pathway invivo in a dose-responsive manner. The upregulation seen at the 0.9 and0.3 mg/kg doses of agonist D is greater than that seen with injectionsof Hh protein at 10 mg/kg/injection, 2×/day.

Lung Branching Assay

E12.5 old ptc-1 (d11) lacZ lungs were harvested and transgenic embryosidentified by lacZ detection using tails. Explants were assembled on 1μm polycarbonate filters (Costar) placed on top of plastic grids(histology embedding chamber) and placed in standard 12-well tissueculture plates filled with lung explant culture medium (DMEM based,additives optimized for the culture of mouse lungs) for 48 hrs, fixed inlacZ fixative, rinsed and stained for lacZ O/N at 37° C.

Results are depicted in FIG. 44. In the control panel on the left, LacZexpression can be observed in the mesenchyme immediately adjacent todistal branching tips, a pattern reflective of endogenous patchedexpression. Treatment with 5 μM of agonist B leads to significantlyincreased reporter gene expression and expansion of the expressiondomain of the transgene, indicative of hedgehog pathway upregulation.The rightmost panel shows the results of treatment with 5 μg/mL of Hhprotein.

Kidney Branching Assay

E13.5 old ptc-1 (d11) lacZ lungs were harvested and transgenic embryosidentified by lacZ detection using tails. Explants were assembled on 1μm polycarbonate filters (Costar) placed on top of plastic grids(histology embedding chamber) and placed in standard 12-well tissueculture plates filled with kidney explant culture medium (DMEM based,additives optimized for the culture of mouse lungs) for 48 hrs, fixed inlacZ fixative, rinsed and stained for lacZ O/N at 37° C.

Results are depicted in FIG. 45. In the control panel on the left, LacZexpression can be observed in the mesenchyme immediately adjacent toproximalmost ureteric epithelium, a pattern reflective of endogenouspatched expression. Treatment with 5 μM of agonist B leads tosignificantly increased reporter gene expression and expansion of theexpression domain of the transgene, indicative of hedgehog pathwayupregulation. Note that the signal remains localized to the mesenchymeand does not expand into the more distally located ureteric and tubularepithelia, indicating that only the mesenchymal cell type(s) respondingto hedgehog signaling in the endogenous situation respond to theagonist, while cell types which usually do not activate this pathway areunaffected by agonist treatment. The rightmost panel shows the resultsof treatment with 5 μg/mL of Hh protein.

Skin Explants

Skin from ptc-lacZ E17.5 pups was excised with a 2 mm skin punch. Thoseskin punches were then cultured for 6 days in control media, or mediaincluding either agonist B or D or Hh protein, or media including bothan agonist and Hh protein. The explants were then stained with X-Galstain. FIG. 46A shows results for agonist B. Treatment with the agonistalone shows greater staining than culturing with a low dose of Hhprotein alone, and treatment with agonist and a low dose of Hh proteinshows staining similar to that of a substantially higher dose of Hhprotein. Analogous results for agonist D are presented in FIG. 46B.

Activity in Human Cells

FIGS. 47A and B compare the activity of subject compounds O and R inmouse reporter cells (TM3 cells with a Gli-Luc reporter construct, asdescribed above) and in human reporter cells (human embryonic palatalmesenchyme (HEPM) cells with a Gli-Luc construct). FIG. 48 shows aquantitative PCR analysis of RNA expressed from the hedgehog target geneGli-1 in human cells treated with the vehicle, Hh protein, and agonistO. The activation of the reporter cell line and the elevated Gli-1message in response to the compounds demonstrate that this agonistfunctions in human cells.

Preparation of Compounds of the Present Invention

a. Illustrative Synthetic Schemes

Exemplary synthesis schemes for generating hedgehog agonists useful inthe methods and compositions of the present invention are shown in FIGS.1-31.

The reaction conditions in the illustrated schemes of FIGS. 1-31 are asfollows:

1) R₁CH₂CN, NaNH₂, toluene

(Arzneim-Forsch, 1990, 40, 11, 1242)

2) H₂SO₄, H₂O, reflux

(Arzneim-Forsch, 1990, 40, 11, 1242)

3) H₂SO₄, EtOH, reflux

(Arzneim-Forsch, 1990, 40, 11, 1242)

4) NaOH, EtOH, reflux

5) (Boc)₂O, 2M NaOH, THF

6) LiHDMS, R₁X, THF

(Merck Patent Applic #WO 96/06609)

7) Pd-C, H₂, MeOH

8) t-BuONO, CuBr, HBr, H₂O

(J. Org. Chem. 1977, 42, 2426)

9) ArB(OH)₂, Pd(PPh₃)₄, Dioxane

(J. Med. Chem. 1996, 39, 217-223)

10) R₁₂(H)C═CR₁₃R₁₄, Pd(OAc)₂, Et₃N, DMF

(Org. React. 1982, 27, 345)

11) Tf₂O, THF

(J. Am. Chem. Soc. 1987, 109, 5478-5486)

12) ArSnBu₃, Pd(PPh₃)₄, Dioxane

(J. Am. Chem. Soc. 1987, 109, 5478-5486)

13) KMnO₄, Py, H₂O

(J. Med. Chem. 1996, 39, 217-223)

14) NaOR₁, THF

15) NaSR₁, THF

16) HNR₁R₁₃, THF

17) HONO, NaBF₄

(Adv. Fluorine Chem. 1965, 4, 1-30)

18) Pd(OAc)₂, NaH, DPPF, PhCH₃, R₁OH

(J. Org. Chem. 1997, 62, 5413-5418)

19) i. R₁X, Et₃N, CH₂Cl₂, ii. R₁₃X

20) SOCl₂, cat DMF

21) CH₂N₂, Et₂O

22) Ag₂O, Na₂CO₃, Na₂S₂O₃, H₂O

(Tetrahedron Lett. 1979, 2667)

23) AgO₂CPh, Et₃N, MeOH

(Org. Syn., 1970, 50, 77; J. Am. Chem. Soc. 1987, 109, 5432)

24) LiOH, THF-MeOH

25) (EtO)₂P(O)CH₂CO₂R, BuLi, THF

26) MeO₂CCH(Br)═P(Ph)₃, benzene

27) KOH or KOtBu

28) Base, X(CH₂)_(n)CO₂R

29) DPPA, Et₃N, toluene

(Synthesis 1985, 220)

30) HONO, H₂O

31) SO₂, CuCl, HCl, H₂O

(Synthesis 1969, 1-10, 6)

32) Lawesson's reagent, toluene

(Tetrahedron Asym. 1996, 7, 12, 3553)

33) R₂M, solvent

34) 30% H₂O₂, glacial CH₃CO₂H

(Helv. Chim. Acta. 1968, 349, 323)

35) triphosgene, CH₂Cl₂

(Tetrahedron Lett., 1996, 37, 8589)

36) i. (EtO)₂P(O)CHLiSO₂Oi-Pr, THF, ii. NaI

37) Ph₃PCH₃I, NaCH₂S(O)CH₃, DMSO

(Synthesis 1987, 498)

38) Br₂, CHCl₃ or other solvent

(Synthesis 1987, 498)

39) BuLi, Bu₃SnCl

40) ClSO₂OTMS, CCl₄

(Chem. Ber. 1995, 128, 575-580)

41) MeOH—HCl, reflux

42) LAH, Et₂O or LiBH₄, EtOH or BH₃-THF

(Tetrahedron Left., 1996, 37, 8589)

43) MsCl, Et₃N, CH₂Cl₂

(Tetrahedron Lett., 1996, 37, 8589)

44) Na₂SO₃, H₂O

(Tetrahedron Lett., 1996, 37, 8589)

45) R₂R₄NH, Et₃N, CH₂Cl₂

46) R₂M, solvent

47) CH₃NH(OCH₃), EDC, HOBt, DIEA, CH₂Cl₂ or DMF

(Tetrahedron Lett, 1981, 22, 3815)

48) MeLi, THF

49) mCPBA, CH₂Cl₂

50) HONO, Cu₂O, Cu(NO₃)₂, H₂O

(J. Org. Chem. 1977, 42, 2053)

51) R₁M, solvent

52) HONO, NaS(S)COEt, H₂O

(Org. Synth. 1947, 27, 81)

53) HSR₂ or HSR₄, CH₂Cl₂

54) i-BuOC(O)Cl, Et₃N, NH₃, THF

55) R₂R₄NH, CH₂Cl₂, NaBH(OAc)₃

56) R₂R₄NH, MeOH/CH₃CO₂H, NaBH₃CN

57) R₂OH, EDC, HOBt, DIEA, CH₂Cl₂ or DMF

58) R₂OH, HBTU, HOBt, DIEA, CH₂Cl₂ or DMF

59) R₂R₄NH, EDC, HOBt, DIEA, CH₂Cl₂ or DMF

60) R₂R₄NH, HBTU, HOBt, DIEA, CH₂Cl₂ or DMF

61) POCl₃, Py, CH₂Cl₂

62) R₂R₄NCO, solvent

63) R₂OC(O)Cl, Et₃N, solvent

64) R₂CO₂H, EDC or HBTU, HOBt, DIEA, CH₂Cl₂ or DMF

65) R₂X, Et₃N, solvent

66) (CH₃S)₂C═N(CN), DMF, EtOH

(J. Med. Chem. 1994, 37, 57-66)

67) R₂SO₂Cl, Et₃N, CH₂Cl₂

68) R₂- or R₃- or R₄CHO, MeOH/CH₃CO₂H, NaBH3CN

(Synthesis 1975, 135-146)

69) Boc(Tr)-D or L-CysOH, HBTU, HOBt, DIEA, CH₂Cl₂ or DMF

70) Boc(Tr)-D or L-CysH, NaBH₃CN, MeOH/CH₃CO₂H

(Synthesis 1975, 135-146)

71) S-Tr-N-Boc cysteinal, ClCH₂CH₂Cl or THF, NaBH(OAc)₃

(J. Org. Chem. 1996, 61, 3849-3862)

72) TFA, CH₂Cl₂, Et₃SiH or (3:1:1) thioanisole/ethanedithiol/DMS

73) TFA, CH₂Cl₂

74) DPPA, Et₃N, toluene, HOCH₂CH₂SiCH₃

(Tetrahedron Lett. 1984, 25, 3515)

75) TBAF, THF

76) Base, TrSH or BnSH

77) Base, R₂X or R₄X

78) R₃NH₂, MeOH/CH₃CO₂H, NaBH₃CN

79) N₂H₄, KOH

80) Pd₂(dba)₃, P(o-tol)₃, RNH₂, NaOtBu, Dioxane, R₁NH₂

(Tetrahedron Lett. 1996, 37, 7181-7184).

81) Cyanamide.

82) Fmoc-Cl, sodium bicarbonate.

83) BnCOCl, sodium carbonate.

84) AllylOCOCl, pyridine.

85) Benzyl bromide, base.

86) Oxalyl chloride, DMSO.

87) RCONH₂.

88) Carbonyldiimidazole, neutral solvents (e.g., DCM, DMF, THF,toluene).

89) Thiocarbonyldiimidazole, neutral solvents (e.g., DCM, DMF, THF,toluene).

90) Cyanogen bromide, neutral solvents (e.g., DCM, DMF, THF, toluene).

91) RCOCl, Triethylamine

92) RNHNH₂, EDC.

93) RO₂CCOCl, Et₃N, DCM.

94) MsOH, Pyridine (J. Het. Chem., 1980, 607.)

95) Base, neutral solvents (e.g., DCM, toluene, THF).

96) H₂NOR, EDC.

97) RCSNH₂.

98) RCOCHBrR, neutral solvents (e.g., DCM, DMF, THF, toluene), (Org.Proc. Prep. Intl., 1992, 24, 127).

99) CH₂N₂, HCl. (Synthesis, 1993, 197).

100) NH₂NHR, neutral solvents (e.g., DCM, DMF, THF, toluene).

101) RSO₂Cl, DMAP. (Tetrahedron Lett., 1993, 34, 2749).

102) Et₃N, RX. (J. Org. Chem., 1990, 55, 6037).

103) NOCl or Cl₂ (J. Org. Chem., 1990, 55, 3916).

104) H₂NOH, neutral solvents (e.g., DCM, DMF, THF, toluene).

105) RCCR, neutral solvents (DCM, THF, Toluene).

106) RCHCHR, neutral solvents (DCM, THF, Toluene).

107) H₂NOH, HCl.

108) Thiocarbonyldiimidazole, SiO₂ or BF₃OEt₂. (J. Med. Chem., 1996, 39,5228).

109) Thiocarbonyldiimidazole, DBU or DBN. (J. Med. Chem., 1996, 39,5228).

110) HNO₂, HCl.

111) ClCH₂CO₂Et (Org. Reactions, 1959, 10, 143).

112) Morpholine enamine (Eur. J. Med. Chem., 1982, 17, 27).

113) RCOCHR′CN

114) RCOCHR′CO₂Et

115) Na₂SO₃

116) H₂NCHRCO₂Et

117) EtO₂CCHRNCO

118) RCNHNH₂.

119) RCOCO₂H, (J. Med. Chem., 1995, 38, 3741).

120) RCHO, KOAc.

121) 2-Fluoronitrobenzene.

122) SnCl₂, EtOH, DMF.

123) RCHO, NaBH₃CN, HOAc.

124) NH₃, MeOH.

125) 2,4,6-Me₃PhSO₂NH₂.

126) Et₂NH, CH₂Cl₂

127) MeOC(O)Cl, Et₃N, CH₂Cl₂

128) R₂NH₂, EDC, HOBT, Et₃N, CH₂Cl₂

129) DBU, PhCH₃

130) BocNHCH(CH₂STr)CH₂NH₂, EDC, HOBT, Et₃N, CH₂Cl₂

131) R₂NHCH₂CO₂Me, HBTU, HOBT, Et₃N, CH₂Cl₂

132) BocNHCH(CH₂STr)CH₂OMs, LiHMDS, THF

133) R₂NHCH₂CO₂Me, NaBH(OAc)₃, ClCH₂CH₂Cl or THF

134) R₂NHCH₂CH(OEt)₂, HBTU, HOBT, Et₃N, CH₂Cl₂

135) NaBH(OAc)₃, ClCH₂CH₂Cl or THF, AcOH.

136) Piperidine, DMF.

137) Pd(Ph₃P)₄, Bu₃SnH.

138) RCO₂H, EDC, HOBT, Et₃N, DCM.

139) RNH₂, neutral solvents.

140) RCHO, NaBH₃CN, HOAc.

141) RNCO, solvent.

142) RCO₂H, EDC or HBTU, HOBt, DIEA, CH₂Cl₂ or DMF.

143) RCOCl, Triethylamine

144) RSO₂Cl, Et₃N, CH₂Cl₂.

145) SnCl₂, EtOH, DMF.

146) RNH₂, EDC, HOBt, DIEA, CH₂Cl₂ or DMF.

147) Dibromoethane, Et₃N, CH₂Cl₂

148) Oxalyl chloride, neutral solvents.

149) LiOH, THF—MeOH.

150) Carbonyldiimidazole, neutral solvents (e.g., DCM, DMF, THF,toluene).

151) RNH₂, Et₃N, CH₂Cl₂.

152) Base, RX.

153) DBU, PhCH₃

154) DPPA, Et₃N, toluene (Synthesis 1985, 220)

155) SOCl₂, cat DMF.

156) ArH, Lewis Acid (AlCl₃, SnCl₄, TiCl₄), CH₂Cl₂.

157) H₂NCHRCO₂Et, neutral solvents.

158) BocHNCHRCO₂H, EDC OR HBTU, HOBt, DIEA, CH₂Cl₂ or DMF.

159) TFA, CH₂Cl₂.

b. Illustrative Preparation of Aryl Subunits

Ary subunits may be functionalized using a wide variety of reactionsknown to those in the art. The chemistry of aromatic and heteroaromaticrings is rich, and only a sampling of useful reactions can be presentedhere. A number of illustrative examples, particularly useful forgenerating the biaryl portion of the subject compounds, are shown below.

c. Illustrative Preparation of Coupling Substrates

Members of the general classes of coupling substrates outlinedabove—arylstannanes, arylboronic acids, aryl triflates and arylhalides—are available from the parent heterocycles. In general, thetransformations required to prepare a coupling substrate are reliableand amenable to scale-up. Illustrative examples are shown below.

Solid Phase Synthesis of Subject Compounds

General:

Washing Protocols

Method 1: water (3×), acetone (2×), N,N-dimethylformamide (3×), water(2×), acetone (1×), N,N-dimethylformamide (3×), water (2×), acetone(3×), methanol (3×), acetone (3×) and methanol (3×);

Method 2: dichloromethane, hexane, N,N-dimethylformamide,dichloromethane, hexane, dichloromethane and hexane;

Method 3: water, N,N-dimethylformamide, water, 1.0 M aqueous sodiumhydroxide solution, water, N-N dimethylformamide, water, 1.0 aqueoussodium hydroxide solution, water, N,N-dimethylformamide,dichloromethane, methanol, dichloromethane, and methanol.

Method 4: N,N-dimethylformamide, dichloromethane, N,N-dimethylformamide,dichloromethane, methanol, dichloromethane, methanol (2×) and ether(2×).

Step A—Preparation of (Nitrophen-4′-yloxycarboxy)benz-4-yloxymethylPolystyrene-(Wang PNP Carbonate Polystyrene) Hydroxybenz-4-yloxymethylpolystyrene (Wang Resin)

Sodium methoxide (233 g, 4.31 mol) was added slowly to a stirred mixtureof chloromethyl polystyrene (2.4 kg, 3.6 mol functionalised loading) and4-hydroxybenzyl alcohol (581 g, 4.68 mol) in N,N-dimethylacetamide (10L) at room temperature under nitrogen. After dilution withN,N-dimethylacetamide (13 L), the mixture was heated at 50° C. for 5 hand then filtered via cannula through a P-ETFE mesh (70 μm). The crudeproduct was washed extensively using the sequence of Method 1, thendried under vacuum at 60° C. to give 2630 g of the title resin.

(Nitrophen-4′-yloxycarboxy)benz-4-yloxymethyl Polystyrene-(Wang PNPCarbonate Polystyrene)

4-Methylmorpholine (660 mL, 6.0 mol) was added dropwise over 2 h to astirred mixture of hydroxybenz-4-yloxymethyl polystyrene (2000 g, 2.5mol functionalised loading) and 4-nitrophenol chloroformate (1209 g, 6.0mol) in dichloromethane (22 L) at (0° C. under nitrogen. The mixture waswarmed gradually to room temperature, stirred overnight and filtered viacannula through a P-ETFE mesh (70 μm). The crude resin was washedextensively using the sequence of Method 2, then dried under vacuum atroom temperature to give 2728 g of a mixture of the title resin and4-methylmorpholine hydrochloride.

Step B—The Preparation of Wang Resin-Bound Diamines General Method (forPiperazine, Homopiperazine and Trans-1,4-diaminocyclohexane)

Crude (nitrophen-4′-yloxycarboxy)benz-4-yloxymethyl polystryrene (1002.5g, ˜0.9 mol functionalised loading) was swollen over 15 min in a 50% v/vmixture of anhydrous dichloromethane and N,N-dimethylformamide (9 L)under nitrogen. N,N-diisopropylamine (626 mL, 5 mol equivalents) and theappropriate diamine (5 mol equivalents) were added and the mixture wasstirred vigorously overnight at room temperature. The mixture wasfiltered through a P-ETFE mesh (70 μm), washed extensively using thesequence of Method 3 and dried under vacuum at 60° C. to give theresin-bound diamine.

Ethylenediamine Bound to Wang Resin

Crude (nitrophen-4′-yloxycarboxy)benz-4-yloxymethyl polystyrene (1002.5g, ˜0.9 mol functionalised loading) was swollen over 15 min indichloromethane (7 L) under nitrogen and treated with ethylenediamine(181 mL, 2.7 mol). The resulting thick, yellow suspension was dilutedwith dichloromethane (2 L) and vigorously stirred overnight at roomtemperature. The mixture was filtered through a P-PETFE mesh (70 μm),washed extensively using the sequence of Method 3 and dried under vacuumat 60° C. to give the title resin-bound diamine.

m-Xylylenediamine Bound to Wang Resin

Crude (nitrophen-4′-yloxycarboxy)benz-4-yloxymethyl polystyrene (1002.5g, ˜0.9 mol functionalised loading) was swollen in tetrahydrofuran (7 L)over 15 min under nitrogen and treated with a solution ofm-xylylenediamine (828 mL, 6.27 mol) in tetrahydrofuran (1 L). Theresulting thick yellow suspension was diluted with dichloromethane (2 L)and vigorously stirred overnight at room temperature. The mixture wasfiltered through a P-ETFE mesh (70 μm), washed extensively using thesequence of Method 3 and dried under vacuum at 60° C. to give theresin-bound diamine.

Step C: Preparation of the Building Block using a Suzuki CouplingProcedure

A suspension of the appropriate aryl bromide (1 equivalent) andpotassium carbonate (2.2 equivalents) in toluene (13 volumes) wasstirred and degassed at room temperature.Tetrakis(triphenylphosphine)palladium(0) (0.01 equivalent) was added andthe reaction vessel evacuated and purged with nitrogen (three times).After 15 min, a degassed solution of 2-methoxy-5-formylphenylboronicacid (1.2 equivalents) in ethanol (6.3 volumes) was added via cannula,then the mixture was heated under reflux and stirred overnight undernitrogen. After cooling, the solid was filtered from solution and washedthoroughly with toluene. The filtrate was evaporated to dryness underreduced pressure to give the crude product. This was triturated withdiethyl ether (5 volumes) and the resulting slurry was filtered, washedwith diethyl ether and dried under vacuum. The biaryl aldehyde wasobtained as a yellow powder.

Step D: Building Block Loading onto Wang Diamine Reductive Alkylation

The appropriate resin (1 equivalent, ˜0.75 mmol functionalised loading)was swollen in a mixture of tetrahydrofuran, trimethylorthoformate anddichloromethane (1:1:1, v/v/v, 10 mL) over 15 min, then gently agitatedand treated with the appropriate aldehyde (2 equivalents). After gentleagitation overnight at room temperature, the resin was filtered, washedthoroughly with tetrahydrofuran and dried under vacuum at 40° C. Thedried resin was then swollen in tetrahydrofuran over 15 min and treatedwith acetic acid (0.12 equivalent) and sodium triacetoxyborohydride (5equivalents). The resin suspension was gently agitated overnight at roomtemperature, then filtered, washed extensively using the sequence ofMethod 4 and dried under vacuum at 60° C.

Acid Chloride Capping

The appropriate resin (1 equivalent) was swollen in dichloromethane (10volumes) over 10 min and treated with the appropriate acid chloride (3equivalents) and N,N-diisopropylethylamine (3 equivalents). The resinsuspension was gently agitated overnight at room temperature, filtered,and washed extensively using the sequence of Method 4 and dried undervacuum at 40° C.

Solution Phase Synthesis of Subject Compounds

The following exemplary scheme illustrates one route through whichhedgehog agonists of the present invention may be prepared. Variationson this exemplary pathway will be readily comprehended and executed bythose of ordinary skill in the art, permitting the preparation of a widerange of compounds that fall within the disclosed general formulae. Thecompound numbers used in this scheme are consistent with the proceduresbelow, and are independent of the compound numbers used elsewhere in theapplication, such as the figures.

Synthesis of 3-Chloro-benzo[b]thiophene-2-carboxylic acid(4′-cyano-6-methoxy-biphenyl-3-ylmethyl)-(4-methylaminocyclohexyl)-amideHydrochloride (7)

(4-Amino-cyclohexyl)-carbamic Acid t-butyl Ester (1)

A solution of di-t-butyl-dicarbonate (12.0 g, 54.7 mmol) andtetrahydrofuran (250 mL) was added slowly under nitrogen over 3 h to asuspension of 1,4-diaminocyclohexane (50.0 g, 0.44 mol) intetrahydrofuran (250 mL) while maintaining the temperature below 10° C.The mixture was allowed to warm to room temperature and subsequentlystirred for 16 hours, then filtered. The filtrate was concentrated invacuo to give a residue. Water (500 mL) was added to the residue,followed by stirring for approximately 15 min, after which the mixturewas filtered and the aqueous layer was extracted with dichloromethane(3×200 mL). The organic extracts were combined and concentrated to givea residue which was dissolved in t-butylmethyl ether (350 mL) and washedwith water (3×50 mL). The t-butylmethyl ether was removed in vacuo togive the title compound 1 (8.1 g, 69%) as a solid: δ_(H) (360 MHz:CDCl₃) 1.06-1.24 (m, 4H), 1.43 (s, 9H), 1.83 (d, 2H), 1.98 (d, 2H),2.56-2.66 (m, 1H), 3.30-3.35 (m, 1H) and 4.31-4.38 (m, 1H).

N-Methyl-cyclohexane-1,4-diamine (2)

Amine 1 (15.0 g, 0.7 mol) was added slowly over 45 min to a 1N solutionof lithium aluminium hydride in THF (450 mL, 0.36 mol) under nitrogen.The mixture was stirred for 30 min at room temperature, then heated atreflux for 5-6 hours under nitrogen. Water (13.2 mL) was added to themixture followed by 15% aqueous sodium hydroxide (13.2 mL), and water(39.7 mL). The mixture was then stirred for 15-30 min. The solid wasfiltered off and washed with t-butylmethyl ether (200 mL),dichloromethane (200 mL), and t-butylmethyl ether (200 mL). The organicextracts were collected, dried (MgSO₄), and filtered. The drying agentwas then washed with dichloromethane and the organic extracts combinedand concentrated in vacuo to give the title compound 2 (7.52 g, 84%) asa pale yellow solid: δ_(H) (360 MHz: CDCl₃) 1.04-1.20 (q, 4H), 1.51 (brs, 3H), 1.80-1.96 (m, 4H), 2.25-2.35 (m, 1H), 2.41 (s, 3H), 2.61-2.72(m, 1H).

(4-Amino-cyclohexyl)-methyl-carbamic Acid t-butyl Ester (3)

Benzaldehyde (12.8 mL, 0.13 mol) was added in a single portion to asolution of N-methylamine 2 (16.2 g, 0.13 mol) and toluene (150 mL)under nitrogen The resulting mixture was heated to reflux usingDean-Stark apparatus for 4 h. After allowing the mixture to cool to roomtemperature, di-t-butyl dicarbonate (27.5 g, 0.13 mol) was added inportions and the mixture stirred for 16 h. The mixture was concentratedin vacuo to leave a yellow oil, to which 1N aqueous potassium hydrogensulfate (90 mL) was added followed by vigorously stirring until TLCindicated the reaction was complete (˜2.5 h). The mixture was extractedinto ether (3×100 mL) and the aqueous layer made alkaline (pH ˜12) withaqueous sodium hydroxide. The aqueous layer was then saturated withsodium chloride and the product extracted into chloroform (3×40 mL). Thecombined extracts were concentrated in vacuo to give the title compound3 (16.3 g, 59%) as a yellow oil: δ_(H) (360 MHz: CDCl₃) 1.11-1.34 (m,5H), 1.45 (s, 9H), 1.66 (br d, 2H), 1.90 (br d, 2H), 2.56-2.66 (m, 1H),2.71 (s, 3H) and 3.98 (br s, 2H).

{4-[(4′-Cyano-6-methoxy-biphenyl-3-ylmethyl)-amino]-cyclohexyl}-methyl-carbamicAcid t-butyl Ester (5)

A solution of amine 3 (5.0 g, 21.92 mmol), aldehyde 4 (5.19 g, 21.92mmol) and trimethyl orthoformate (50 ml) were stirred at roomtemperature under nitrogen for 16 h. Sodium triacetoxyborohydride (6.5g, 30.7 mmol) was then added portion wise and the mixture stirred atroom temperature until the reaction was complete, as determined by LC-MSanalysis. Water was added carefully and mixture stirred for a 5 min,followed by separation of the layers. The trimethyl orthoformate layerwas poured onto 1N aqueous potassium hydrogen sulfate (100 mL) andstirrred for 15 min. The precipitated solid was filtered, washed withwater (50 mL), cold tributyl methyl ether (3×30 mL). The washedprecipitate was then suspended in dichloromethane (150 mL) to whichsaturated aqueous sodium hydrogen carbonate (50 mL) was added and the pHmade alkaline (pH ˜10) whilst maintaining vigorous stirring. Thedichloromethane layer was washed with water, and brine, and the organicextract was dried (MgSO₄) and concentrated in vacuo to give the titlecompound 5 (6.9 g, 69%) as an off white solid: δ_(H) (360 MHz: CDCl₃)1.18-1.31 (m, 4H), 1.43 (s, 9H), 1.68 (d, 2H), 2.01 (d, 2H), 2.44-2.56(m, 1H), 2.69 (s, 3H), 3.76 (s, 2H), 3.80 (s, 3H), 6.92 (d, 1H), 7.24(s, 1H), 7.29 (d, 1H), 7.61 (d, 2H) and 7.66 (d, 2H).

{4-[(3-Chloro-benzo[b]thiophene-2-carbonyl)-(4′-cyano-6-methoxy-biphenyl-3-ylmethyl)-amino]-cyclohexyl}-methyl-carbamicAcid t-butyl Ester (6)

N,N-Diisopropylethylamine (2.1 mL, 12.1 mmol) was added to a solution ofamine 5 (2.2 g, 4.9 mmol), 3-chlorobenzo[b]thiophene-2-carbonyl chloride(1.3 g, 5.86 mmol) and anhydrous dichloromethane (22 mL), with stirringunder argon. Once all of the starting material had been consumed asmonitored by TLC (˜2.5 hours), the mixture was washed with water,saturated aqueous sodium hydrogen carbonate, and brine. The organiclayer was dried (MgSO₄), and concentrated in vacuo. The yellow residuewas then purified by silica-gel chromatography using hexane/ethylacetate 3:1 to give the title compound 6 (2.93 g, 93%) as a pale yellowsolid.

3-Chloro-benzo[b]thiophene-2-carboxylic Acid(4′-cyano-6-methoxy-biphenyl-3-ylmethyl)-(4-methylamino-cyclohexyl)-amideHydrochloride (7)

Concentrated hydrochloric acid (27.5 mL) was added to a solution ofcompound 6 (11.0 g, 17.1 mmol) and ethanol (82.5 mL). The mixture wasstirred until reaction was complete as monitored by TLC. The mixture wasconcentrated in vacuo and dichloromethane added and concentrated again,this was repeated until a solid was obtained. The solid was thenslurried with t-butylmethyl ether (30 mL), filtered, and the organiclayer dried (MgSO₄) and concentrated in vacuo to give the title compound7 (10.0 g, 99%): δ_(H) (360 MHz: DMSO, 70° C.) 1.30-1.50 (m, 2H), 1.85(br s, 4H), 2.12 (br d, 2H), 2.48 (s, 3H), 2.91 (br t, 1H), 3.81 (s,3H), 3.87 (br s, 1H), 4.71 (s, 2H), 7.14 (d, 1H), 7.29 (s, 1H), 7.40 (d,1H), 7.57-7.68 (m, 4H), 7.80-7.90 (m, 3H), 8.09 (d, 1H), 8.82 (br s,2H).

All publications and patents cited herein are hereby incorporated byreference in their entirety.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. compound represented in general formula (X):

wherein, as valence and stability permit, Ar represents a substituted orunsubstituted aryl or heteroaryl ring; X is selected from —C(═O)—,—C(═S)—, —S(O₂)—, —S(O)—, and a methylene group optionally substitutedwith 1-2 lower alkyls; Y is absent for each occurrence; Z is absent orrepresents a substituted or unsubstituted aryl, carbocyclyl,heterocyclyl, or heteroaryl ring, or a lower alkyl, nitro, cyano, orhalogen substituent; R represents, independently for each occurrence, Hor substituted or unsubstituted lower alkyl; Cy′ represents a3-chloro-benzo(b)thien-2-yl, 3-fluoro-benzo(b)thien-2-yl, or3-methyl-benzo(b)thien-2-yl, wherein the benzo ring is substituted withfrom 1-4 substituents selected from halogen, nitro, cyano, methyl, andethyl; M represents, independently for each occurrence, a substituted orunsubstituted methylene group, or two M taken together representsubstituted or unsubstituted ethene or ethyne, wherein some or alloccurrences of M in M_(j) form all or part of a cyclic structure; jrepresents an integer from 2 to 7; i represents 0 for all occurrencesexcept in the sequence N—M_(i)—Y—Ar, where i represents 1; and krepresents
 0. 2. The compound of claim 1, wherein M_(j) includes acycloalkyl ring having 5-7 ring atoms.
 3. The compound of claim 2,wherein NR₂ represents NHMe.
 4. The compound of claim 1, wherein Cy is acyclohexyl ring substituted with a primary or secondary amine.
 5. Thecompound of claim 4, wherein the amine is a methylamino group.
 6. Thecompound of claim 1, wherein the benzo ring is substituted with from 1-4substituents selected from halogen, and methyl.
 7. The compound of claim6, wherein the benzo ring is a 1,4-difluorobenzene ring.
 8. A compoundrepresented in general formula (XI):

wherein, as valence and stability permit, Ar represents a substituted orunsubstituted aryl or heteroaryl ring; X is selected from —C(═O)—,—C(═S)—, —S(O₂)—, —S(O)—, and a methylene group optionally substitutedwith 1-2 lower alkyls; Y is absent for each occurrence; Z is absent orrepresents a substituted or unsubstituted aryl, carbocyclyl,heterocyclyl, or heteroaryl ring, or a lower alkyl, nitro, cyano, orhalogen substituent; R represents, independently for each occurrence, Hor substituted or unsubstituted lower alkyl; Cy′ represents a3-chloro-benzo(b)thien-2-yl, 3-fluoro-benzo(b)thien-2-yl, or3-methyl-benzo(b)thien-2-yl, wherein the benzo ring is substituted withfrom 1-4 substituents selected from halogen, nitro, cyano, methyl, andethyl; M represents, independently for each occurrence, a substituted orunsubstituted methylene group, or two adjacent M taken togetherrepresent substituted or unsubstituted ethene or ethyne; Cy represents asubstituted or unsubstituted aryl, heterocyclyl, heteroaryl, orcycloalkyl, including polycyclic groups; i represents 0 for alloccurrences except in the sequence N—M_(i)—Y—Ar, where i represents 1;and k represents
 0. 9. The compound of claim 8, wherein Cy is acyclohexyl ring substituted with a primary or secondary amine.
 10. Thecompound of claim 9, wherein the amine is a methylamino group.
 11. Thecompound of claim 8, wherein the benzo ring is substituted with from 1-4substituents selected from halogen, and methyl.
 12. The compound ofclaim 11, wherein the benzo ring is a 1,4-difluorobenzene ring.
 13. Thecompound of claim 8, wherein X is selected from —C(═O)—, —C(═S)—, and—S(O₂)—.
 14. The compound of claim 13, wherein Cy represents asubstituted or unsubstituted cycloalkyl group.
 15. The compound of claim13, wherein NR₂ represents a secondary amine.
 16. The compound of claim13, wherein the benzo ring is substituted with from 1-4 substituentsselected from halogen and methyl.
 17. The compound of claim 16, whereineach R independently represents H or a lower alkyl.
 18. The compound ofclaim 15, wherein the benzo ring is substituted with from 1-4substituents selected from halogen and methyl.
 19. The compound of claim18, wherein each R independently represents H or a lower alkyl.
 20. Thecompound of claim 13, wherein Cy represents a substituted orunsubstituted cycloalkyl group.
 21. The compound of claim 20, whereinNR₂ represents a secondary amine.
 22. The compound of claim 20, whereinthe benzo ring is substituted with from 1-4 substituents selected fromhalogen and methyl.
 23. The compound of claim 21, wherein X is selectedfrom —C(═O)—, —C(═S)—, and —S(O₂)—.
 24. The compound of claim 21,wherein each R independently represents H or a lower alkyl.
 25. Thecompound of claim 22, wherein each R independently represents H or alower alkyl.
 26. The compound of claim 22, wherein X is selected from—C(═O)—, —C(═S)—, and —S(O₂)—.
 27. The compound of claim 8, wherein NR₂represents a secondary amine.
 28. The compound of claim 27, wherein thebenzo ring is substituted with from 1-4 substituents selected fromhalogen, and methyl.
 29. The compound of claim 27, wherein each Rindependently represents H or a lower alkyl.
 30. The compound of claim28, wherein Cy represents a substituted or unsubstituted cycloalkylgroup.